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. 2012 Jan;18(1):101-11.
doi: 10.1002/ibd.21744. Epub 2011 May 6.

Loss of downregulated in adenoma (DRA) impairs mucosal HCO3(-) secretion in murine ileocolonic inflammation

Fang Xiao  1 , Marina Juric, Junhua Li, Brigitte Riederer, Sunil Yeruva, Anurag Kumar Singh, Lifei Zheng, Silke Glage, George Kollias, Pradeep Dudeja, De-An Tian, Gang Xu, Jinxia Zhu, Oliver Bachmann, Ursula Seidler
Affiliations

Loss of downregulated in adenoma (DRA) impairs mucosal HCO3(-) secretion in murine ileocolonic inflammation

Fang Xiao et al. Inflamm Bowel Dis. 2012 Jan.

Abstract

Background: Ileocolonic luminal pH has been reported to be abnormally low in inflammatory bowel disease (IBD) patients, and one of the causative factors may be reduced epithelial HCO(3)(-) secretory rate (J(HCO3)(-)). Disturbances in J(HCO3)(-) may occur due to inflammation-induced changes in the crypt and villous architecture, or due to the effect of proinflammatory cytokines on epithelial ion transporters VSports手机版. .

Methods: To discriminate between these possibilities, the tumor necrosis factor alpha (TNF-α) overexpressing (TNF(+/ΔARE)) mouse model was chosen, which displays high proinflammatory cytokine levels in both ileum and colon, but develops only mild colonic histopathology and diarrhea. HCO(3)(-) secretion, mRNA expression, immunohistochemistry, and fluid absorptive capacity were measured in ileal and mid-colonic mucosa of TNF(+/ΔARE) and wildtype (WT) (TNF(+/+)) mice in Ussing chambers, and in anesthetized mice in vivo V体育安卓版. .

Results: The high basal J(HCO3)(-) observed in WT ileal and mid-colonic mucosa were luminal Cl(-) -dependent and strongly decreased in TNF(+/ΔARE) mice V体育ios版. Downregulated in adenoma (DRA) mRNA and protein expression was strongly decreased in TNF(+/ΔARE) ileocolon, whereas cystic fibrosis transmembrane conductance regulator (CFTR), Na(+) /H(+) exchanger 3 (NHE3), Na(+) /HCO(3)(-) cotransporter (NBC), and epithelial sodium channel (ENaC) expression was not significantly altered. This indicates that the severe defect in ileocolonic J(HCO3)(-) was due to DRA downregulation. Fluid absorption was severely depressed in the ileum but only mildly affected in the mid-distal colon, preventing the development of overt diarrhea. .

Conclusions: Even mild ileocolonic inflammation may result in a decrease of epithelial HCO(3)(-) secretion, which may contribute to alterations in surface pH, intestinal flora, and mucus barrier properties VSports最新版本. .

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"V体育官网入口" Figures

Figure 1
Figure 1
Manifestation of chronic inflammation in TNF+/ΔARE mice distal-ileum and mid-colon. (A) Increase of mRNA expression for the proinflammatory cytokines TNF-α (left panel), IFN-γ (middle panel), and IL-1β (right panel) in the distal ileum and the mid-colon of TNF+/ΔARE (□) mice in relation to the TNF+/+ controls (■). mRNA expression in the TNF+/+ was set to 1. Villin was used as control gene. *P < 0.05 versus TNF+/+. n = 4. (B) Distal ileal histology, with marked villus blunting and inflammatory cell infiltration in TNF+/ΔARE mice (c,d) compared to TNF+/+ (a,b). (C) Mid-colonic histology, with submucosal inflammatory cell infiltration in TNF+/ΔARE mice (c,d) compared to TNF+/+ (a,b) and slight crypt and goblet cell hypertrophy. Scale bars = 200 μm.
Figure 2
Figure 2
JHCO3− and Isc in different segments of murine ileocolon. (A) Time course of JHCO3− and (B) Isc in the presence and absence of luminal Cl− and after forskolin stimulation across TNF+/+ distal ileal, proximal, and mid-colonic mucosa. (A) Ileal and mid-colonic mucosa display high basal JHCO3−, which were strongly reduced by removal of Cl− from the luminal perfusate and therefore to a large part mediated by Cl−/HCO3− exchange. In the proximal colon (first 1–1.5 cm of colon), basal JHCO3− was low and Cl− independent. (B) No significant differences in basal and Fsk-stimulated Isc (ΔIsc) among these segments of the intestine. The arrows indicate the addition of forskolin (Fsk, 10 μM) to the serosal bath. n = 5–9.
Figure 3
Figure 3
Basal and forskolin-stimulated JHCO3− and Isc across TNF+/ΔARE and TNF+/+ littermate distal ileal mucosa. (A) JHCO3− was assessed in the presence and absence of luminal CI−, as well as after stimulation with forskolin (10−5M), in TNF+/ΔARE (□) and TNF+/+ (■) distal ileal mucosa. Left panel: Time course. Middle panel: JHCO3− in the presence (NaCl) and absence (Na-Gluconate) of Cl− in the luminal bath. Right panel: forskolin-stimulated ΔJHCO3−. (B) Time course (left panel), basal Isc in the presence and absence of luminal Cl− (middle panel), and forskolin-stimulated Isc (ΔIsc, right panel) across TNF+/ΔARE (□) and TNF+/+ (■) distal ileal mucosa. *P < 0.05 versus TNF+/+. n = 7–8.
Figure 4
Figure 4
Basal and forskolin-stimulated JHCO3− and Isc across TNF+/ΔARE and TNF+/+ mid-colonic mucosa. (A) JHCO3− was assessed in the presence and absence of luminal CI−, as well as after stimulation with forskolin (10−5M), in TNF+/ΔARE (□) and TNF+/+ (■) mid-colonic mucosa. Left panel: Time course. Middle panel: JHCO3− in the presence (NaCl) and absence (Na-Gluconate) of Cl− in the luminal bath. Right panel: forskolin stimulated ΔJHCO3−. (B) Time course (left panel), basal Isc in the presence and absence of luminal Cl− (middle panel), and ΔIsc (right panel) in TNF+/ΔARE (□) and TNF+/+ (■) mid-colonic mucosa. *P < 0.05 versus TNF+/+. n = 5.
Figure 5
Figure 5
JHCO3− in luminally perfused distal ileum, proximal-mid, and mid-distal colon in anesthetized TNF+/ΔARE and TNF+/+ mice. (A) Time course (left panel), basal JHCO3− in the presence and absence of luminal Cl− (middle panel) and forskolin-stimulated JHCO3− (ΔJHCO3−, right panel) in perfused distal ileum of TNF+/ΔARE (□) and TNF+/+ (■) mice. Both the basal JHCO3− (middle panel) as well as the forskolin-induced ΔJHCO3− (right panel) are significantly reduced in TNF+/ΔARE ileum. (B) In inflamed proximal-mid (left panel) and mid-distal (right panel) colon, basal JHCO3− are significantly decreased in TNF+/ΔARE (□) compared to TNF+/+ (■) mice in vivo. *P < 0.05 versus TNF+/+. n = 3–5.
Figure 6
Figure 6
Significant decrease of DRA expression in the inflamed ileal and colonic mucosa. (A) mRNA expression for DRA and PAT-1 in the distal ileum of TNF+/ΔARE (□) was significantly decreased compared to TNF+/+ (■) ileum. The mRNA expression levels in the TNF+/+ ileum was set to 1. The qPCR was performed in both groups against villin (left panel), actin (middle panel), and RPS9 (right panel) as control genes. **P < 0.01, ***P < 0.001 versus TNF+/+ mice. n = 4. (B) mRNA expression for DRA, CFTR, and NHE3 in the mid-colon of TNF+/ΔARE (□) compared to TNF+/+ (■) mid-colon. The qPCR was performed in both groups against cytokeratin 18 (left panel), villin (middle panel), and RPS9 (right panel) as control genes. *P < 0.05 versus TNF+/+ mice. n = 4. (C) Immunofluorescence of DRA (green) in TNF+/ΔARE and TNF+/+ mice mid-colon. DRA fluorescence is decreased in the apical membrane of colonic enterocytes of TNF+/ΔARE (b) compared to TNF+/+ (a). Scale bars = 10 μm. A representative of three different experiments is shown.
Figure 7
Figure 7
Fluid absorption in the distal ileum and mid-distal colon of anesthetized TNF+/ΔARE and TNF+/+ mice. (A) Fluid absorptive rates were strongly decreased in distal ileum of anesthetized TNF+/ΔARE compared to TNF+/+ mice, and the fluid secretory response was also strongly reduced. *P < 0.05. n = 5. (B) In contrast, fluid absorptive rates in the mid-distal colon of TNF+/ΔARE mice were not significantly reduced compared to TNF+/+ mice, and the ENaC inhibitor amiloride had even a slightly stronger inhibitory effect in the TNF+/ΔARE colon, indicative of intact ENaC function. *P < 0.05. n = 4.
Figure 8
Figure 8
Intact ENaC activity and normal ENaC mRNA expression in TNF+/ΔARE mice distal colon. (A) Left panel shows time course of amiloride-sensitive Isc in distal colonic mucosa of TNF+/ΔARE and TNF+/+ mice that were either fed with normal or low salt chow. Right panel: Differences in Isc were measured before and after apical addition of amiloride (10 μM), and the amiloride-sensitive Isc is displayed. *P < 0.05 versus normal food groups. n = 4. (B) ENaC subunit mRNA expression in the distal colon. Left panel shows mRNA expression of ENaC α, β, and γ subunit in distal colon of TNF+/+ mice on either normal or after 3 weeks of low Na+ diet, demonstrating upregulation of α and γ subunits. Middle panel: No significant difference in ENaC subunit expression was observed in TNF+/ΔARE and TNF+/+ distal colon mice under standard chow. Right panel: After 3 weeks of low Na+ diet, ENaC subunit expression was not significantly different in TNF+/ΔARE and TNF+/+ distal colon, indicating intact stimulation of ENaC by salt restriction. In (B), the relative mRNA expression of the α-subunit in the TNF+/+ colon was set to 1. Expression of ENaC subunits was standardized to that of the epithelial marker cytokeratin 18. *P < 0.05 versus TNF+/+. n = 3–6.
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