1. Volume 126, Issue 7, Pages 1711-1720 (June 2004)
Cytokine-dependent transcriptional down-regulation of epithelial sodium channel in ulcerative colitis 
Salah Amasheh, Christian Barmeyer, Corinna Swanette Koch, Shida Tavalali, Joachim Mankertz, Hans-Jörg Epple, Maren Melanie Gehring, Peter Florian, Anton-Josef Kroesen, Martin Zeitz, Michael Fromm, Jörg-Dieter Schulzke 
Gastroenterology 
Volume 126, Issue 7, Pages (June 2004)
DOI: /j.gastro

2. Figure 1
Effect of aldosterone (3 · 10−9 mol/L) on ENaC. Time course of active electrogenic sodium transport in human sigmoid colon induced by the mineralocorticoid aldosterone in vitro. In control tissue, a steady increase of short-circuit current (ISC) was induced by incubation with aldosterone. Maximum JNa was determined after 8 hours by the drop of short-circuit current induced by 100 μmol/L of the ENaC inhibitor amiloride (n = 8). UC tissues did not reveal significant JNa.
Gastroenterology  , DOI: ( /j.gastro )

3. Figure 2
ISC in ulcerative colitis. Comparison of ISC before and after addition of aldosterone. In control colon, 3 nmol/L aldosterone induced a strong increase in ISC, which dropped again after addition of amiloride (amil). This amiloride-sensitive drop in ISC (JNa) was 6.9 ± 1.2 μmol · h−1 · cm−2. In inflamed tissues, JNa was diminished to 0.06 ± 0.04 μmol · h−1 · cm−2. Data are means ± SEM of n = 8 (control tissue) and n = 7 (UC tissue) experiments for each group (∗P < 0.05).
Gastroenterology  , DOI: ( /j.gastro )

4. Figure 3
Induction of ENaC mRNA in ulcerative colitis: Northern blots. In control tissue, 3 nmol/L aldosterone induced a strong increase of the β- and γ-ENaC mRNA, whereas α-ENaC remained stable. In ulcerative colitis, α-ENaC mRNA was still detectable. Furthermore, aldosterone-dependent induction of the β- and γ-ENaC mRNA was abolished in UC. Representative blots of n = 6 or 7 experiments for each group.
Gastroenterology  , DOI: ( /j.gastro )

5. Figure 4
Induction of ENaC subunits in ulcerative colitis. (A) Western blots. (B) Densitometry of Western blots. (C ) Immunostainings. (D) Western blot analysis of the mineralocorticoid receptor. (A and B) Changes of α-, β-, and γ-ENaC subunits were analyzed by immunoblotting. Subunit-specific signals were confirmed by blocking experiments employing the respective peptides used for immunization. A strong increase of β- and γ-ENaC signals was observed exclusively in the membrane fraction of control tissues incubated with aldosterone but not in inflamed tissues. No significant changes were detectable for α-ENaC signals, which were still present also in UC (blots of n = 4, pooled tissues). Each antibody labeled a single band at 72–76 kilodaltons. (C) Distributions of α-, β-, and, γ-ENaC in sigmoid colon of controls and inflamed tissues after 8 hours of incubation with aldosterone. α-ENaC was constitutively expressed in both control and inflamed colon at the surface epithelium, whereas β- and γ-ENaC expression was limited to controls after incubation with aldosterone (stainings performed on adjacent sections of n = 4, respectively; representative data shown). (D) Expression of the mineralocorticoid receptor. MR-specific signals were detectable in both controls and UC tissues at 116 kilodaltons (blots of n = 4 each, pooled tissues).
Gastroenterology  , DOI: ( /j.gastro )

6. Figure 5
Induction of ENaC after exposure to proinflammatory cytokines. (A) Electrophysiology. (B) Northern blots. (C) Densitometry of Northern blots. In control tissues, the amiloride-sensitive drop in ISC (JNa) as a measure of ENaC induction was reduced to 47% ± 8% when TNF-α (100 ng/mL) and IFN-γ (1000 U/mL) were present (∗P < 0.05). After 8-hour incubation with aldosterone, a strong increase of the β- and γ-ENaC mRNA was detected, which was dramatically reduced in the presence of the 2 proinflammatory cytokines. Densitometry revealed an aldosterone-induced increase of the β- and γ-ENaC signals to 363% ± 61% and 526% ± 39%, respectively, in the absence vs. no significant change (104% ± 14% and 92% ± 7%, respectively) in the presence of TNF-α and IFN-γ. In addition, no significant changes of α-ENaC signals were detected (percentage of controls, n = 4, respectively).
Gastroenterology  , DOI: ( /j.gastro )

7. Figure 6
Promoter analysis. (A) Relative activity of the human ENaC β-subunit promoter compared with the untreated controls in the presence of 2500 U/mL TNF-α (26% ± 5%, n = 3, ∗∗P < 0.01) or 100 U/mL IFN-γ (79% ± 12%, n = 3, ns). (B) Relative activity of the human ENaC γ-subunit promoter compared with the untreated controls in the presence of 2500 U/mL TNF-α (36% ± 4%, n = 3, ∗∗P < 0.01) or 100 U/mL IFN-γ (110% ± 9%, n = 3, ns).
Gastroenterology  , DOI: ( /j.gastro )