Mechanism of down regulation of Na-H exchanger-2 in experimental colitis

Abstract

Background: The Na-H exchanger [NHE] performs an electroneutral uptake of NaCl and water from the lumen of the gastrointestinal tract. There are several distinct NHE isoforms, some of which show an altered expression in the inflammatory bowel diseases (IBD). In this study, we examined a role of NHE-2 in experimental colitis.

Methods: Colitis was induced in male Sprague-Dawley rats by intra-rectal administration of trinitrobenzenesulphonic acid (TNBS). On day 6 post-TNBS, the animals were sacrificed, colonic and ileal segments were taken out, cleaned with phosphate buffered saline and used in this study.

Results: There was a significant decrease in the level of NHE-2 protein as measured by ECL western blot analysis and confocal immunofluorescence microscopy. The levels of NHE-2 mRNA and heteronuclear RNA measured by an end-point RT-PCR and a real time PCR were also decreased significantly in the inflamed colon. However, there was no change in the level of NHE-2 protein in response to in vitro TNF-α treatment of uninflamed rat colonic segment. These changes were selective and localized to the colon as actin, an internal control, remained unchanged. Confocal immunofluorescence microscopy revealed co-localization of NHE-2 and NHE-3 in the brush borders of colonic epithelial cells. Inflamed colon showed a significant increase in myeloperoxidase activity and colon hypertrophy. In addition, there was a significant decrease in body weight and goblet cells' mucin staining in the TNBS treated colon. These changes were not conspicuous in the non-inflamed ileum.

Conclusions: These findings demonstrate suppression of NHE-2 expression on the brush borders in the colonic epithelial cells which is regulated transcriptionally. However a role of TNF-α in the regulation of NHE-2 is discounted in the present model of colitis. This decrease in the NHE-2 expression will lead to a loss of electrolyte and water uptake thus contributing to the symptoms associated with IBD.

Fig 1. Bar diagram showing myeloperoxidase (MPO) activity as units per mg tissue in the colon (closed bars), and ileum (open bars) taken from the uninflamed (control) and inflamed rat colon on day 6 post-TNBS.

Data are mean±SEM (n = 20). *Indicates significance p<0.05 between the control and TNBS.

Fig 2

Upper panel: Shown are the percent changes in the body weight (BW) on day 6 post-TNBS as compared with their initial body weights at day 0. Data are mean±SEM (n = 20). *Indicates significance p<0.05 with respect to their initial weights at day 0. Lower panel: Bar diagram showing colon weight (mg) per cm length (hypertrophy) of the uninflamed (control) and inflamed rat colon on day 6 post-TNBS. Data are mean±SEM (n = 20). *Indicates significance p<0.05 versus non colitis control.

Fig 3. Representative picture (n = 9) showing H&E stained colon sections (A = Control, B = Colitis).

Muscle hypertrophy and infiltration of inflammatory cells are evident. Goblet cells staining with Alcian blue dye of uninflamed colon [C] and inflamed colon [D]. A reduction of mucin expression is evident in the inflamed colon.

Fig 4. A representative confocal fluorescence microscopy picture showing expression of NHE-2 and NHE-3 and their colocalization in the epithelial brush borders of the control non inflamed and inflamed colon.

Nuclei have been stained with DAPI. Magnification was 40x.

Fig 5. A representative ECL western blot picture showing expression of NHE-2 and actin proteins (n = 9) in three (1–3) controls and three (1–3) TNBS colitisin colonic (upper panel), and ileal (lower panel) segments.

Fig 6. Bar diagrams showing the level of NHE-2 protein expression as a ratio NHE-2:actin in the control and TNBS–treated rat colon (closed bars) and Ileum (open bars).

Data are mean±SEM (n = 9), *Indicates significance p<0.05) with respect to the controls.

Fig 7. A representative (n = 9) confocal microscopy picture showing the levels of NHE-2 protein in the non colitic control and TNBS inflamed colon.

Arrow indicates the location of NHE-2 expression (red) and blue dye indicates staining of the nuclei (DAPI). Magnification 40x (upper panel). Lower panel: Quantitative data shown as mean±SEM (n = 9). From each section 3–4 fields were selected for quantitation. *Indicates significance P<0.05 with respect to controls (lower panel).

Fig 8. Agarose gel picture showing quality of total RNA extracted from the control (C) and colitis (T) colonic tissues used in this study.

The 28S and 18S bands are ribosomal RNA (upper panel). Expression of NHE-2 mRNA (300 bp) and the competitive control (CC, 175 bp) in the control (C) and TNBS inflamed (T) colon using the end-point RT-PCR method (lower panel).

Fig 9. Bar diagram showing expression levels of mRNA as measured by the end-point RT-PCR (closed bars) and a SYBR green real time PCR (open bars).

Quantitative expression level measured as ratios (NHE-2:CC) are shown. Data are mean±SEM (n = 9). *Indicates P<0.05 versus controls.

Fig 10. Bar diagram showing expression level of hnRNA as obtained from RT-PCR.

Data are mean±SEM (n = 9). *Indicates P<0.05versus controls.