1. Volume 134, Issue 5, Pages 1436-1447 (May 2008)
Altered ENaC Expression Leads to Impaired Sodium Absorption in the Noninflamed Intestine in Crohn's Disease 
Sebastian Zeissig, Theresa Bergann, Anja Fromm, Christian Bojarski, Frank Heller, Ute Guenther, Martin Zeitz, Michael Fromm, Jörg– Dieter Schulzke 
Gastroenterology 
Volume 134, Issue 5, Pages (May 2008)
DOI: /j.gastro
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2. Figure 1
Electrogenic sodium absorption is impaired in the macroscopically noninflamed sigmoid colon of patients with small intestinal Crohn's disease (CD). (A and C) Time course of electrogenic sodium transport and transepithelial resistance (Rt) in biopsy specimens of sigmoid colon from control and CD patients. Aldosterone (3 · 10−9 mol/L) was added at the beginning of the experiment, and electrogenic sodium absorption via ENaC was determined by addition of 10−4 mol/L amiloride after 8 hours. Rt was constantly registered to confirm viability of specimens and showed only a modest decrease over time because of induction of short-circuit current (Isc), which was reversed by final addition of amiloride. (B and D) Electrogenic sodium absorption and transepithelial resistance of CD (n = 8 patients) patients and control (n = 8 patients). Experiments were performed as described above. Electrogenic sodium absorption, expressed as amiloride-sensitive Isc (ΔIsc), was determined after 8 hours of aldosterone stimulation. Transepithelial resistance was determined 30 minutes after the beginning of the experiment. Mean ± SEM are indicated. (E and F) Original impedance locus plots as determined in biopsy samples of sigmoid colon from control (E) and macroscopically noninflamed CD (F). Zreal gives the ohmic component and Zimaginary the reactive component of the complex impedance. Intersections between the semicircle and x-axis at low and high frequencies represent Rt and Rsub, respectively. Rt minus Rsub equals Re.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
ENaC in aldosterone-treated and fresh frozen sigmoid colon of control and macroscopically noninflamed Crohn's disease (CD). (A) ENaC subunit mRNA in sigmoid colon after aldosterone stimulation. Biopsy specimens of sigmoid colon were mounted into Ussing chambers and treated with aldosterone (3 · 10−9 mol/L, 8 hours) or left untreated. Fold induction represents the relative induction of α-, β, and γ-ENaC mRNA in aldosterone-treated biopsy specimens over that of untreated biopsy specimens. The results are mean ± SEM of n = 6 controls and n = 7 CD patients. **P < .01 compared with controls. (B and C) Subcellular localization of γ-ENaC in controls (B) and CD (C). Biopsy specimens were stimulated in Ussing chambers for 8 hours with 3 · 10−9 mol/L aldosterone. Cryosections of biopsy specimens were stained with anti-γ-ENaC antibody (red), anti-E-cadherin antibody (green) to visualize the lateral cell membrane, and DAPI (blue) to counterstain nuclei. Bar indicates 20 μm. Lumen indicates the intestinal lumen. All images were taken using the same instrument settings. One representative experiment is shown; 2 additional independent experiments gave comparable results. (D) ENaC subunit mRNA in fresh frozen sigmoid colon. Biopsy specimens of sigmoid colon were either frozen directly after extraction or mounted into Ussing chambers and incubated in the absence of aldosterone for 8 hours. Real-time PCR was performed as described in A and is expressed as fold induction of fresh frozen biopsy specimens over biopsy specimens incubated in the absence of aldosterone. γ-ENaC mRNA levels in biopsy specimens incubated in the absence of aldosterone were low but always detectable. In contrast, γ-ENaC could not be detected in any sample of fresh frozen CD colon (0/4), which is indicated as N.D. (not detectable). The results are mean ± SEM of n = 8 controls and n = 4 CD patients. **P < .01 compared with CD.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Extracellular signal-regulated kinase (ERK) is involved in suppression of electrogenic sodium absorption in rat colon. (A) Immunoblots of phosphorylated p44/42 (ERK1/2), p38, and JNK. Rat colon was incubated in Ussing chambers with TNF-α (100 ng/mL) for indicated periods, and MAPK were detected by specific antibodies. One representative experiment is shown; 3 additional independent experiments gave comparable results. (B–D) Rat colon was incubated for 1 hour with MAPK inhibitors (10 μmol/L) followed by incubation with TNF-α (100 ng/mL) for 6 hours. Subsequently, 3 · 10−9 mol/L aldosterone was added for 8 hours, followed by addition of amiloride. The amiloride-sensitive Isc reflects ENaC-dependent sodium flux and is expressed as flux of monovalent cations (μmol · h−1 · cm−2). Data are mean ± SEM of n = 5 specimens from independent animals per condition. ##P < .01 vs aldosterone treatment; **P < .01 vs TNF-α/aldosterone-treated specimens.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Extracellular signal-regulated kinase (ERK) is involved in TNF-α-mediated suppression of ENaC transcription in rat colon. (A–C) Rat distal colon was incubated in Ussing chambers with U0126 (10 μmol/L) for 1 hour and/or TNF-α (100 ng/mL) for another 6 hours, followed by aldosterone treatment (3 · 10−9 mol/L) for 8 hours. After determination of amiloride-sensitive Isc, real-time PCR was performed. Fold induction represents the relative induction of α-, β, and γ-ENaC mRNA over that of nonaldosterone treated colon. The results are mean ± SEM of n = 6 tissues from independent animals per condition. **P < .01 compared with aldosterone treatment. (D–F) Correlation between the amiloride-sensitive Isc and the relative mRNA induction of α-ENaC (D), β-ENaC (E), and γ-ENaC (F) as obtained from specimens shown in A–C. R2 indicates the correlation coefficient.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

6. Figure 5
Distribution of γ-ENaC in rat distal colon. Rat distal colon was mounted in Ussing chambers and was left untreated (A) or stimulated with 3 · 10−9 mol/L aldosterone for 8 hours (B). In C, tissues were incubated with TNF-α (100 ng/mL) for 6 hours before addition of aldosterone. In D, U0126 (10 μmol/L) was added 1 hour before addition of aldosterone. In E, incubation with U0126 (1 hour) was followed by addition of TNF-α for 6 hours and aldosterone for a further 8 hours. Cryosections were stained with anti-γ-ENaC antibody (red), anti-E-cadherin antibody (green) to visualize the lateral cell membrane, and DAPI (blue) to counterstain nuclei. Bar indicates 20 μm. All images were taken with the same instrument settings. One representative experiment is shown; 2 additional independent experiments gave comparable results.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

7. Figure 6
Inhibition of MEK1/2 restores electrogenic sodium absorption in sigmoid colon of macroscopically noninflamed Crohn's disease. Aldosterone (3 · 10−9 mol/L) and U0126 (10 μmol/L) were added at the beginning of the experiment as indicated. Electrogenic sodium absorption was determined as amiloride-sensitive short-circuit current (ΔIsc) after 8 hours of aldosterone stimulation. Mean ± SEM are indicated (n = 4). **P < .01 vs aldosterone treatment alone.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions