Aldosterone Reduces Crypt Colon Permeability during Low-Sodium Adaptation

Fluid and electrolyte absorption by colonic crypts depends on the transport properties of crypt cellular and paracellular routes and of the pericryptal sheath. As a low-Na⁺ diet increases aldosterone and angiotensin II secretion, either hormone could affect absorption. Control and adrenalectomized (ADX) Sprague-Dawley rats were kept at a high-NaCl (HS) diet and then switched to low-NaCl (LS) diet for 3 days. Aldosterone or angiotensin II plasma concentrations were maintained using implanted osmotic mini-pumps. The extracellular Na⁺ concentration in isolated rat distal colonic mucosa was determined by confocal microscopy using a low-affinity Na⁺-sensitive fluorescent dye (Sodium red, and Na⁺-insensitive BODIPY) bound to polystyrene beads. Crypt permeability to FITC-labelled dextran (10 kDa) was monitored by its rate of escape from the crypt lumen into the pericryptal space. Mucosal ion permeability was estimated by transepithelial electrical resistance (TER) and amiloride-sensitive short-circuit current (SCC). The epithelial Na⁺ channel, ENaC, was determined by immunolocalization. LS diet decreased crypt wall permeability to dextran by 10-fold and doubled TER. Following ADX, aldosterone decreased crypt wall dextran permeability, increased TER, increased Na⁺ accumulation in the pericryptal sheath and ENaC expression even in HS. Infusion of angiotensin II to ADX rats did not reverse the effects of aldosterone deprivation. These findings indicate that aldosterone alone is responsible for both the increase in Na⁺ absorption and the decreased paracellular and pericryptal sheath permeability.     

In vivo fluorescence measurement of Na+ concentration in the pericryptal space of mouse descending colon

A methodinvolving surgical exposure of the colonic mucosa, fluorescent dyeaddition, and confocal microscopy has been developed for monitoringcolonic crypt function in vivo in mice. Na⁺ concentrationin the extracellular pericryptal space of descending colon was measuredusing a low-affinity Na⁺-sensitive fluorescent indicatorconsisting of an Na⁺-sensitive chromophore (sodium red) andan Na⁺-insensitive chromophore (Bodipy-fl) immobilized on200-nm-diameter polystyrene beads. The Na⁺ indicator beadsaccumulated in the pericryptal spaces surrounding the colonic cryptsafter a 1-h exposure of the colonic luminal surface to the beadsuspension. Na⁺ concentration ([Na⁺]) in thepericryptal space was 491 ± 62 mM (n = 4). Aftera 70-min exposure to amiloride (0.25 mM), pericryptal[Na⁺] was reduced to 152 ± 21 mM. Blockage of thecrypt lumen with mineral oil droplets reduced pericryptal[Na⁺] to 204 ± 44 mM. Exposure of the colonicmucosa to FITC-dextran (4.5 kDa) led to rapid accumulation of the dyeinto the crypt lumen with a half time of 19.8 ± 1.0 s, whichwas increased to 77.9 ± 6.0 s after amiloride treatment.These results establish an in vivo fluorescence method to measurecolonic crypt function and provide direct evidence for accumulation ofa hypertonic absorbate in the pericryptal space of descending colon.The pericryptal space represents the first example of a hypertonicextracellular compartment in mammals that is not created by acountercurrent amplification mechanism.

     

Effects of decorin on the expression of α-smooth muscle actin in a human myofibroblast cell line

Myofibroblasts are metabolically and morphologically distinctive fibroblasts expressing α-smooth muscle actin (α-SMA), and their activation plays a key role in development of the fibrotic response. In an activated state, myofibroblasts cease to proliferate and start to synthesize large amounts of extracellular component proteins. The expression of α-SMA correlates with the activation of myofibroblasts. Decorin, a member of the small leucine-rich proteoglycan gene family, has been implicated in the negative control of cell proliferation primarily by upregulating the expression of p21, a potent inhibitor of cyclin-dependent kinase. In order to examine the effect of decorin on myofibroblast cell growth, we rendered a human lung myofibroblast cell line, MRC-5, quiescent by either cell–cell contact or serum starvation, and examined the relationship between decorin and α-SMA expression in these cells. The expression of decorin in cells made quiescent by serum starvation was lower than that in cells made quiescent by cell–cell contact. In contrast, the expression of α-SMA in cells made quiescent by cell–cell contact was lower than that in cells made quiescent by serum starvation. Furthermore, forced expression of decorin was accompanied by a suppression of α-SMA expression, whereas knocking down of decorin expression by RNA interference increased the expression of α-SMA.     

Development of Na+ transport in the chicken colon

To evaluate the developmental changes in colonic Na⁺ transport, Na, K-ATPase activity and the sensitivity of the short-circuit current to amiloride were investigated. The amiloride-sensitive short-circuit current which represents the electrogenic, amiloride-sensitive Na⁺ transport through Na⁺ channels, was not present in chicken embryos but rose significantly after hatching in chicks which were kept on a low-salt diet. Amiloride-sensitive short-circuit current increased gradually but the plateau was not reached during the first 15 days of life. Drinking of 0.9% NaCl totally inhibited the induction of amiloride-sensitive Na⁺ transport. Na⁺, K⁺-ATPase activity increased during development but was not influenced by changes in salt intake. Na⁺ transport in chicken colon therefore undergoes profound developmental changes. The increase of Na⁺ transport refleets not only the adaptation of colonocytes to low salt intake but also the maturation of Na⁺ absorption in colon. The possible role of aldosterone in the adaptation to low-salt intake is discussed.Abbreviations LS low-salt - HS high-salt - I _sc short-circuit current     

Regulation of adrenal atrial natriuretic hormone receptor subtypes.

Regulation of atrial natriuretic hormone (ANH) receptor binding and aldosterone suppression was studied in isolated adrenal glomerulosa cells from rats fed a high-salt (HS) or low-salt (LS) diet for 3 days. In plasma of HS rats, aldosterone levels were 5 times lower and immunoreactive ANH two times higher than in LS rats. Competitive binding studies showed the same affinity for human atrial natriuretic hormone (hANH) in both pools of cells, but receptor density was 50% higher on LS cells. A linear ANH analog that binds to non-guanylate-cyclase-coupled receptors did not show increased binding to LS cells. Cyclic GMP production in response to hANH was identical in both groups. The aldosterone-inhibitory effect of hANH on both groups of basal and angiotensin II-stimulated cells was also identical. Thus a short-term high-salt diet causes decreased density of ANH receptors in glomerulosa cells without changing biological activity of ANH. These results suggest that dietary salt content changes the number of ANH receptors and that non-guanylate-cyclase-coupled receptors contain at least two classes of receptors.     

TGFβ and bFGF synthesis and localization in Dupuytren''s disease (nodular palmar fibromatosis) relative to cellular activity, myofibroblast phenotype and oncofetal variants of fibronectin

Summary Nodular palmar fibromatosis is a self-limited proliferation of fibro-/myofibroblasts associated with growth factor synthesis and abundant fibronectin extracellular matrix deposition. bFGF and TGFβ are potent modulators of fibro-/myofibroblast proliferation and differentiation. Moreover,in vitro investigations evidenced a TGFβ1-dependent regulation of alternative splicing of fibronectin mRNA. To investigate a possible implication of these growth factors in the tissue formation process of palmar fibromatosis, TGFβ1/2 and bFGF synthesis, as well as TGFβ1/3 and bFGF tissue distribution, is demonstrated by RNAin situ hybridization and/or immunohistochemistry in relation to myofibroblast phenotype development (α-smooth muscle actin, desmin immunohistochemistry), expression of different fibronectin isoforms (ED-A⁺, ED-B⁺ and oncofetal glycosylated fibronectin immunohistochemistry, fibronectin RNAin situ hybridization) and cellular activity (cyclin RNAin situ hybridization, Ki-67 immunolabelling). The myofibroblast phenotype (α-smooth muscle actin, desmin), the growth factor synthesis (TGFβ1 and 2, bFGF), fibronectin matrix synthesis (RNAin situ hybridization with cDNA) and ED-A⁺, ED-B⁺ and oncofetal glycosylated fibronectin immunostaining are exclusively localized in the active proliferative nodules (Ki-67 immunolabelling and cyclin mRNA demonstration). Whereas the growth factor synthesis is restricted to the proliferative areas of the fibromatosis only, TGFβ1, TGFβ3 and bFGF proteins can also be detected immunohistochemically with a lower intensity in the surrounding aponeurotic tissue. The spatial correlation of myofibroblast phenotype, TGFβ and bFGF synthesis and the occurrence of the oncofetal molecular fibronectin variants (ED-B⁺ and oncofetal glycosylated fibronectin) in the active proliferative fibromatosis nodules suggests a pathogentic role of these growth factors and matrix components in the tumorous tissue formation process. The presence of the bFGF and TGFβ1/3 proteins in fibroblasts neighbouring the proliferative nodules may point to a recruitment of quiescent aponeurotic fibroblasts in the fibromatous tissue formation process.     

Rapid Effects of Aldosterone on Sodium-Hydrogen Exchange in Isolated Colonic Crypts

Aldosterone plays a central role in the homeostatic regulation of extracellular fluid volume by stimulating transepithelial electrolyte transport. These effects involve binding to an intracellular receptor, modification of genomic events and protein synthesis. Rapid cellular responses to steroid hormones have been observed in a variety of nonepithelial tissues. The term ``nongenomic' has been proposed for these fast steroid responses since they are unaffected by inhibitors of protein synthesis. We hypothesized that colonic crypts, recently demonstrated to absorb fluid, would respond rapidly to aldosterone. Cytoplasmic pH changes in crypts loaded with a pH-sensitive, fluorescent dye (BCECF) were recorded with confocal laser imaging. An intracellular alkalization of colonic crypts was observed within one minute of aldosterone application that was inhibited by ethylisopropylamiloride or the absence of extracellular sodium, yet unaffected by inhibitors of protein synthesis. The genesis of this rapid and distinct steroid action involves a signal transduction pathway that involves G proteins, protein kinase C, and prostaglandins. We have identified, by real-time imaging, a nongenomic upregulation of sodium-hydrogen exchange in colonic crypts by aldosterone that occurs independent of the traditional receptor. This distinct, rapid onset effect of aldosterone on epithelial ion transport has major implications for our understanding of fluid and electrolyte homeostasis in health and disease. Received: 27 October 1998/Revised: 23 March 1999     

A low-salt diet facilitates Cl secretion in hen lower intestine

Summary The regulation of sodium and chloride transport in hen coprodeum by mineralocorticoids was investigated with isolated epithelia under short-circuit conditions. Unidirectional fluxes of Na and Cl were measured by isotopes and modulated by amiloride, theophylline and bumetanide. Hens were maintained either on low-NaCl diet (LS) or on high-NaCl diet (HS). Plasma aldosterone (PA) levels of these groups were measured with radioimmunoassay. A group of HS hens received injections of aldosterone on a 6-hr schedule before experiments. Another group of LS hens was resalinated, and experiments carried out on a 24-hr interval.Salt deprivation stimulated PA levels ninefold, compared to HS hens. Na absorption was stimulated according to previous reports. Electrogenic Cl secretion was elicited by theophylline and partially inhibited by bumetanide. Modulation of PA levels by diet, resalination or aldosterone injection changed the magnitude of electrogenic Cl secretion in parallel between 0.5 eq/cmau2 · hr (HS) and 4 eq/cm² · hr (LS), with pronounced alteration in tissue resistance.The results demonstrate a new action of aldosterone which besides stimulating Na absorption also directly or indirectly elicits Cl secretion. Evidence is presented for a hormonal adaptation of chloride transport in this epithelium. There was a morphological change of the apical plasma membrane and further experiments will have to clarify the exact cellular nature of this process.     

Induction of the myofibroblast phenotype following elastolytic injury to mouse lung

The repair of alveolar structures following endotracheal administration of porcine pancreatic elastase (PPE) to mice involves the coordinated deposition of new matrix elements. We determined the induction of the myofibroblast phenotype following elastolytic injury to mouse lung by examining the expression of α-smooth muscle actin (α-SMA) by immunohistochemistry. We also examined elastin and α1(I) collagen mRNA expression by in situ hybridization. Changes in airspace dimensions were assessed by determining mean linear intercept. In untreated mice, α-SMA was localized to vascular structures and large airways, with no detectable expression in alveolar units. PPE induced α-SMA expression in damaged areas surrounding large vessels, in septal remnants, and in the opening ring of alveolar ducts. Elastin and α1(I) collagen mRNA expression were up-regulated in residual alveolar structures and septal walls. PPE dose-response studies indicated that α1(I) collagen and elastin mRNA expression were not induced in areas of normal lung adjacent to damaged lung. The administration of low dose PPE resulted in increased α-SMA protein and elastin mRNA expression in the cells comprising the opening ring of alveolar ducts. Our data suggest that repair mechanisms following elastolytic injury are confined to overtly damaged alveolar structures and involve the induction of the myofibroblast phenotype.     

Female gender-specific inhibition of KCNQ1 channels and chloride secretion by 17beta-estradiol in rat distal colonic crypts

The estrogen sex steroid 17beta-estradiol rapidly inhibits secretagogue-stimulated cAMP-dependent Cl(-) secretion in the female rat distal colonic crypt by the inhibition of basolateral K(+) channels. In Ussing chamber studies, both the anti-secretory response and inhibition of basolateral K(+) current was shown to be attenuated by pretreatment with rottlerin, a PKCdelta-specific inhibitor. In whole cell patch-clamp analysis, 17beta-estradiol inhibited a chromanol 293B-sensitive KCNQ1 channel current in isolated female rat distal colonic crypts. Estrogen had no effect on KCNQ1 channel currents in colonic crypts isolated from male rats. Female distal colonic crypts expressed a significantly higher amount of PKCdelta in comparison to male tissue. PKCdelta and PKA were activated at 5 min in response to 17beta-estradiol in female distal colonic crypts only. Both PKCdelta- and PKA-associated with the KCNQ1 channel in response to 17beta-estradiol in female distal colonic crypts, and no associations were observed in crypts from males. PKA activation, association with KCNQ1, and phosphorylation of the channel were regulated by PKCdelta as the responses were blocked by pretreatment with rottlerin. Taken together, our experiments have identified the molecular targets underlying the anti-secretory response to estrogen involving the inhibition of KCNQ1 channel activity via PKCdelta- and PKA-dependent signaling pathways. This is a novel gender-specific mechanism of regulation of an ion channel by estrogen. The anti-secretory response described in this study provides molecular insights whereby estrogen causes fluid retention effects in the female during periods of high circulating plasma estrogen levels.     

Periostin is expressed in pericryptal fibroblasts and cancer-associated fibroblasts in the colon.

Periostin is a unique extracellular matrix protein, deposition of which is enhanced by mechanical stress and the tissue repair process. Its significance in normal and neoplastic colon has not been fully clarified yet. Using immunohistochemistry and immunoelectron microscopy with a highly specific monoclonal antibody, periostin deposition was observed in close proximity to pericryptal fibroblasts of colonic crypts. The pericryptal pattern of periostin deposition was decreased in adenoma and adenocarcinoma, preceding the decrease of the number of pericryptal fibroblasts. Periostin immunoreactivity appeared again at the invasive front of the carcinoma and increased along the appearance of cancer-associated fibroblasts. ISH showed periostin signals in cancer-associated fibroblasts but not in cancer cells. Ki-67-positive epithelial cells were significantly decreased in the colonic crypts of periostin-/- mice (approximately 0.6-fold) compared with periostin+/+ mice. In three-dimensional co-culture within type I collagen gel, both colony size and number of human colon cancer cell line HCT116 cells were significantly larger ( approximately 1.5-fold) when cultured with fibroblasts derived from periostin+/+ mice or periostin-transfected NIH3T3 cells than with those from periostin-/- mice or periostin-non-producing NIH3T3 cells, respectively. Periostin is secreted by pericryptal and cancer-associated fibroblasts in the colon, both of which support the growth of epithelial components.     

Differential influence of butyrate concentration on proximal and distal colonic mucosa in rats born germ-free and associated with a strain of Clostridium paraputrificum

In vivo influence of butyrate in colonic mucosa was studied using a model of gnotobiotic rats monoassociated with a Clostridium paraputrificum. Rats were fed a diet containing increasing amounts of non-digestible carbohydrates, the fermentation of which led to modulated amounts of butyrate in the large intestine. In the proximal colon, the increase in the butyrate concentration alters crypt depth and the number of mucus-containing cells; the increase in butyrate was highly correlated with the number of neutral-mucin-containing cells. Conversely, in the distal colon, no relation was found between the increase in butyrate concentration and crypt depth or number of mucin-containing cells. In both the proximal and distal colon, the mitotic index remained unchanged. In conclusion, in vivo production of physiological quantities of butyrate had a trophic effect on proximal colonic mucosa, but did not influence the distal epithelium.     

Pathways for K<Superscript>+</Superscript> Efflux in Isolated Surface and Crypt Colonic Cells. Activation by Calcium

K⁺-conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring ⁸⁶Rb efflux. In crypt cells, basal K⁺ efflux (rate constant: 0.24 ± 0.044 min⁻¹, span: 24 ± 1.3%) was inhibited by 30 mM TEA and 5 mM Ba²⁺ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin, iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 μM) stimulated K⁺ efflux, increasing the rate constant to 0.65 ± 0.007 min⁻¹ and the span to 83 ± 3.2%. Ionomycin-induced K⁺ efflux was inhibited by clotrimazole (IC₅₀ of 25 ± 0.4 μM) and charybdotoxin (IC₅₀ of 65 ± 5.0 nM) and was insensitive to TEA, Ba²⁺, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca²⁺-activated intermediate-conductance K⁺ channels (IK_ca). Absence of extracellular Ca²⁺ did neither affect basal nor ionomycin-induced K⁺ efflux. However, intracellular Ca²⁺ depletion totally inhibited the ionomycin-induced K⁺ efflux, indicating that the activation of these K⁺ channels mainly depends on intracellular calcium liberation. K⁺ efflux was stimulated by intracellular Ca²⁺ with an EC₅₀ of 1.1 ± 0.04 μM. In surface cells, K⁺ efflux (rate constant: 0.17 ± 0.027 min⁻¹; span: 25 ± 3.4%) was insensitive to TEA and Ba²⁺. However, ionomycin induced K⁺ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present IK_Ca channels but only crypt cells have TEA- and Ba²⁺-sensitive conductive pathways, which would determine their participation in colonic K⁺ secretion.     

Rabbit distal colon epithelium: III. Ca2+-activated K+ channels in basolateral plasma membrane vesicles of surface and crypt cells

Summary In the mammalian distal colon, the surface epithelium is responsible for electrolyte absorption, while the crypts are the site of secretion. This study examines the properties of electrical potential-driven⁸⁶Rb⁺ fluxes through K⁺ channels in basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon epithelium. We show that Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels are present in both surface and crypt cell derived vesicles with half-maximal activation at 5×10^–7 m free Ca²⁺. This suggests an important role of cytoplasmic Ca²⁺ in the regulation of the bidirectional ion fluxes in the colon epithelium.The properties of K⁺ channels in the surface cell membrane fraction differ from those of the channels in the crypt cell derived membranes. The peptide toxin apamin inhibits Ca²⁺-activated K⁺ channels exclusively in surface cell vesicles, while charybdotoxin inhibits predominantely in the crypt cell membrane fraction. Titrations with H⁺ and tetraethylammonium show that both high-and low-sensitive⁸⁶Rb⁺ flux components are present in surface cell vesicles, while the high-sensitive component is absent in the crypt cell membrane fraction. The Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels can be solubilized in CHAPS and reconstituted into phospholipid vesicles. This is an essential step for further characterization of channel properties and for identification of the channel proteins in purification procedures.     

Role of protein kinase C in aldosterone-induced non-genomic inhibition of basolateral potassium channels in human colonic crypts

Aldosterone produces rapid, non-genomic, inhibition of basolateral intermediate conductance K(+) (IK(Ca)) channels in human colonic crypt cells but the intracellular second messengers involved are unclear. We therefore evaluated the role of protein kinase C (PKC) in aldosterone's non-genomic inhibitory effect on basolateral IK(Ca) channels in crypt cells from normal human sigmoid colon. Patch clamp studies revealed that in cell-attached patches, IK(Ca) channel activity decreased progressively to 38+/-8% (P<0.001) of the basal value 10 min after the addition of 1 nmol/L aldosterone, and decreased further to 23+/-6% (P<0.02) of the basal value 5 min after increasing the aldosterone concentration to 10 nmol/L. Pre-incubation of crypts with 1 micromol/L chelerythrine chloride or 1 micromol/L G? 6976 (PKC inhibitors) prevented the inhibitory effect of aldosterone. Conversely, channel activity decreased to 60+/-9% (P<0.02) of the basal value 10 min after the addition of 500 nmol/L PMA (a PKC activator), whereas 4alpha-PMA (an inactive ester) had no effect. When aldosterone (10 nmol/L) and PMA were added together, IK(Ca) channel activity was inhibited to the same extent as with aldosterone alone. These results indicate that aldosterone's non-genomic inhibitory effect on the macroscopic basolateral K(+) conductance in human colonic crypts reflects PKC-mediated inhibition of IK(Ca) channels.     

PROLIFERATION KINETICS IN EPITHELIUM OF GUINEA-PIG COLON

Autoradiographs of crypts of guinea-pig colon labelled with ³H-thymidine were quantitatively analysed. The analysis involved: (a) comparison of proliferation parameters in crypts situated in two well-defined regions of colonic folds, i.e. in the top and the basal parts of the fold, and (b) comparison of proliferation parameters in the lower and in the upper parts of an individual crypt. The results provide evidence that: (1) Crypts situated in the top region of folds are significantly longer than those situated in the basal region. (2) The duration of cell cycle and its phases is similar in the crypts of the two regions examined. (3) The rate of ³H-thymidine uptake is higher in the middle than in the initial and final parts of the S phase, and its pattern is the same for the crypts of the two regions of the fold as well as for the parts of the individual crypt. (4) The proliferating pool size is larger in crypts situated in the top than in the basal region of the fold. (5) The proliferating pool size is larger in the lower parts of crypts from each region than in the upper parts. Control of cellular proliferation in the crypt epithelium is considered to operate separately at the level of the individual crypt and at the level of a group of crypts situated in a given region of the colonic wall.     

Effects of adrenal steroids on Na transport in the lower intestine (Coprodeum) of the hen

Summary The influence of adrenal steroids on sodium transport in hen coprodeum was investigated by electrophysiological methods. Laying hens were maintained on low-NaCl diet (LS), or on high-NaCl diet (HS). HS hens were pretreated with aldosterone (128 g/kg) or dexamethasone (1 mg/kg) before experiment. A group of LS hens received spironolactone (70 or 160 mg/kg, for three days). The effects of these dietary and hormonal manipulations on the amiloride-sensitive part of the short-circuit current were examined. This part is in excellent agreement with the net Na flux, and therefore a direct electrical measurement for Na transport. After depolarizing the basolateral membrane potential with a high K concentration, the apical Na permeability and the intracellular Na activity were investigated by currentvoltage relations for the different experimental conditions.Plasma aldosterone concentrations (PA) were low in HS hens, dexamethasone-treated HS hens and spironolactonetreated LS hens (<70pm). In contrast LS hens and aldosteronetreated HS hens had a PA concentration of 596±70 and 583±172pm, respectively. LS diet (chronic stimulation) had the largest stimulatory effect on Na transport and apical Na permeability. Hormone-treated animals had three- to fourfold lower values. Spironolactone supply in LS hens decreased Na transport and apical Na permeability about 50%.The results provide evidence that both mineralo- and glucocorticoids stimulate Na transport in this tissue by increasing the apical Na permeability. Quantitative differences between acute and chronic stimulation reveal a secondary slower adaptation in apical membrane properties.     

Rise in Extracellular Fluid Volume During High Sodium Depends on BMI in Healthy Men

A high sodium (HS) intake is associated to increased cardiovascular and renal risk, especially in overweight subjects. We hypothesized that abnormal sodium and fluid handling is involved, independent of hypertension or insulin resistance. Therefore, we studied the relation between BMI and sodium‐induced changes in extracellular fluid volume (ECFV; distribution volume of ¹²⁵I‐iothalamate) in 78 healthy men, not selected for BMI. A total of 78 subjects with a median BMI of 22.5 (range: 19.2–33.9 kg/m²) were studied after 1 week on a low sodium (LS) diet (50 mmol Na⁺/d) and after 1 week on HS (200 mmol Na⁺/d). The change from LS to HS resulted in an increase in ECFV of 1.2 ± 1.8 l. Individual changes in ECFV were correlated to BMI (r = 0.361, P < 0.01). Furthermore, in response to HS, a higher BMI was associated to a higher rise in filtered load of sodium (FL_Na+ = [Na⁺] × GFR, r = 0.281, P < 0.05). Thus, a shift to HS leads to a larger rise in ECFV in healthy subjects with higher BMI, associated with an elevated FL_Na+ during HS. Although no hypertension occurred in these healthy subjects, our data provide a potential explanation for the interaction of sodium intake and BMI on cardiovascular and renal risk. Exaggerated fluid retention may be an early pathogenic factor in the cardiorenal complications of overweight.     

Smad3 contributes to positioning of proliferating cells in colonic crypts by inducing EphB receptor protein expression

Deficiency of Smad3, an intracellular mediator of TGF-β, was shown to significantly accelerate re-epithelialization of the colonic mucosa. This study was performed to investigate the molecular mechanisms by which Smad3 controls colonic epithelial cell proliferation and crypt formation. Smad3^ex8/ex8 C57BL/6 mice were used in this study and wild-type littermates served as controls. The number of proliferating cells in the isolated colonic epithelium of Smad3^−/− mice was significantly increased compared to that in wild-type littermates. Protein levels of the cell cycle inhibitors p21 and p27 were significantly decreased, while that of c-Myc was increased in the isolated colonic epithelium from Smad3^−/− mice. In the colonic tissue of wild-type mice, cell proliferation was restricted to the bottom of the crypts in accordance with nuclear β-catenin staining, whereas proliferating cells were located throughout the crypts in Smad3^−/− mice in accordance with nuclear β-catenin staining, suggesting that Smad3 is essential for locating proliferating cells at the bottom of the colonic crypts. Notably, in Smad3^−/− mice, there was loss of EphB2 and EphB3 receptor protein expression, critical regulators of proliferating cell positioning, while EphB receptor protein expression was confirmed at the bottom of the colonic crypts in wild-type mice. These observations indicated that disturbance of the EphB/ephrin B system brings about mispositioning of proliferating cells in the colonic crypts of Smad3^−/− mice. In conclusion, Smad3 is essential for controlling number and positioning of proliferating cells in the colonic crypts and contributes to formation of a “proliferative zone” at the bottom of colonic crypts in the normal colon.     

Epithelial vs myofibroblast differentiation in immortal rat lung cell lines—modulating effects of bleomycin

Two alveolar epithelial cell lines R3/1 and L2 were screened by immunocytochemical and RT-PCR analysis of epithelial and mesenchymal/contractile marker proteins. R3/1 and L2 cells were tested for their sensitivity to bleomycin (BLM), an anticancer drug, which is proposed to induce changes in lung cell differentiation. Both epithelial cell lines exhibited a mixed phenotype consisting of epithelial (E-cadherin, aquaporin-5 and cytokeratin 8) and myofibroblast-like (vimentin, α-SMA and caveolin-3) properties suggesting that the cell lines are arrested in vitro at a certain developmental stage during epithelial–mesenchymal transition (EMT). BLM treatment of R3/1 cells resulted in a partial reversal of this process modifying the cells in an epithelial direction, e.g., upregulation of E-cadherin, aquaporin-5 and other lung epithelial antigens at the mRNA and protein level. L2 cells showed similar alterations following BLM exposure. Immunohistochemical investigation of lung tissue from two different animal models of BLM-induced fibrosis (mouse and rat), revealed no signs of EMT, e.g., myofibroblastic differentiation of alveolar epithelial cells in situ. Immunohistological analysis of tissue samples of the rat model showed a heterogeneous population of myofibroblasts (α-SMA⁺/caveolin-3⁺, α-SMA^-/caveolin-3⁺, and α-SMA⁺/caveolin-3⁻). These results suggest that BLM, on one hand, induces fibrosis and on the other hand possibly suppresses EMT during fibrogenesis.