Ileal Bile Acid Transporter Protein Research Articles

Orthotopic transplantation of intestinal mucosal organoids in rodents

Orthotopic transplantation of intestinal mucosal organoids that contain putative mucosal stem cells serves as an important step toward implementing intestinal gene therapy and treatment for malabsorption syndromes in animals and humans. We hypothesized that intestinal mucosal organoids can be transplanted along the axis of the small bowel giving rise to a neomucosa expressing proteins of its donor origin. Epithelial organoids were harvested from neonatal mice or rat small intestine with the use of a combination of enzymatic digestion with dispase and collagenase, and gravity sedimentation. In adult syngeneic recipients, a 7-cm segment of midjejunum was isolated, leaving its vascular pedicle intact. The remaining proximal and distal segments were anastomosed to restore intestinal continuity. The isolated segments were randomly subjected to surgical or chemical mucosectomy with a chelator solution for 30, 45, or 60 minutes and then compared. Histologic examination was used to confirm the presence of enterocytes, goblet cells, enteroendocrine cells, and Paneth cells in the neomucosal segments. To confirm the presence of ileal bile acid transporter (IBAT) gene message and function, we measured sodium-dependent bile acid uptake and IBAT-messenger RNA. Immunohistochemical examination using anti-IBAT antibodies was performed to demonstrate the expression of IBAT in the neomucosal segments. Experiments were repeated in a murine model transgenic for the green fluorescent protein to verify donor origin of the engrafted mucosa expressing IBAT. The area of peak IBAT function was found to be located in the terminal ileum. Organoid units harvested from this region were capable of generating a small-bowel neoileal mucosa after being seeded into the jejunum. This mucosa was histologically confirmed to differentiate into all 4 intestinal lineages and to express IBAT signal, confirming its donor-derived origin. Optimal engraftment of mucosa expressing the IBAT protein was found in isolated jejunal segments debrided for 45 minutes. Sodium-dependent bile acid uptake was 5-fold higher in the neoileum, compared with the jejunum. IBAT-mRNA levels in the neoileum were 18-100-fold higher than those in the jejunum. Areas of green fluorescent protein-positive mucosa stained positively with anti-IBAT antibody in adjacent sections, suggesting that the regenerated mucosa is from transplanted ileal stem cells. Orthotopic transplantation of epithelial organoids containing ileal stem cells was used to generate a neoileal mucosa that expressed all 4 intestinal lineages along with a new zone of active bile acid uptake and IBAT expression in a recipient jejunal segment.

Treatment of Bile Acid Malabsorption Using Ileal Stem Cell Transplantation

We hypothesized that ileal stem cell clusters transplanted into a segment of jejunum can be used to treat bile acid malabsorption. In adult Lewis rats, a 15-cm segment of jejunum was isolated with its blood circulation left intact and partially stripped of enterocytes using luminal high-velocity perfusions with 3mmol/L ethylenediamine tetra-acetic acid solutions. Continuity was restored by anastomosing the proximal and distal gut. Ileal stem cell clusters were harvested from neonatal Lewis rats and transplanted into the stripped segments to generate a "neoileum." After 4weeks, recipients underwent resection of the native ileum, and the isolated neoileum was anastomosed in its place. After an additional 4weeks, a 48-hour stool collection was performed. The engrafted segment was harvested for taurocholate uptake studies, ileal bile acid transporter (IBAT) protein by immunohistomorphometry, and IBAT mRNA quantitation by reverse transcription polymerease chain reaction. Data were analyzed by ANOVA/t-test. Rats undergoing ileectomy, jejunectomy, or sham operations served as controls. Total bile acid loss in the stool was markedly lower in rats with a neoileum compared with rats with an ileectomy (p < 0.001). Total taurocholate uptake was notably increased in the neoileum compared with the jejunum (p < 0.001). IBAT protein signal intensity was considerably higher in the neoileum compared with jejunum (p < 0.001). IBAT mRNA amounts in the neoileal group were comparable with those in normal rat ileum and were considerably higher (p = 0.003) than in the jejunum. Ileal stem cell clusters were used to establish a new zone of bile acid uptake and IBAT expression in a jejunal segment. This neoileum eliminated loss of bile acids in the stool after ileectomy. This is the first time that transplantation of intestinal stem cell clusters has been shown to correct a clinical malabsorption syndrome.

Treatment of bile acid malabsorption using ileal stem cell transplantation

Purpose: To determine if ileal stem cells transplanted into a segment of jejunum can be used to treat bile acid malabsorption. Methods: In adult Lewis rats, a 15-cm segment of jejunum was isolated with its blood circulation left intact and partially stripped of enterocytes using luminal high-velocity perfusions with 3 mM EDTA solutions. Continuity was restored by anastomosing the proximal and distal gut. Ileal stem cell clusters were harvested from neo-natal Lewis rats and transplanted into these segments. After 4 weeks, rats underwent an ileectomy and the isolated segment was anastomosed in its place. After an additional 4 weeks, a 48 hour stool collection was performed. The engrafted segment was harvested for taurocholate uptake studies, ileal bile acid transporter (IBAT) protein (by immunohistomorphometry) and IBAT mRNA quantitation (by RT-PCR). Data was analyzed by ANOVA/t-test. Rats undergoing ileectomy, jejunectomy or sham operation served as controls. Results: Total bile acid loss in the stool was significantly lower in rats with a neo-ileum compared to rats with an ileectomy (p < 0.004; Fig. 1). Na+-dependent taurocholate uptake was significantly increased in the neo-ileum compared to the jejunum (p < 0.001). IBAT protein signal intensity was significantly higher in the neo-ileum compared to jejunum (p < 0.001). IBAT mRNA amounts were significantly higher (p < 0.004) than in the jejunum however comparable to the ileum. Conclusion: Ileal stem cells were used to establish a new zone of active bile acid uptake and IBAT expression in a jejunal segment. This neo-ileum eliminated loss of bile acids in the stool after ileectomy. This is the first time intestinal stem cell transplantation has been shown to correct a clinical malabsorption syndrome.

Identification of Optimal Harvest Sites of Ileal Stem Cells for Treatment of Bile Acid Malabsorption in a Dog Model

Ileal mucosal stem cells expressing the sodium-dependent ileal bile acid transporter (IBAT) have been successfully transplanted into the jejunum of rodents in projects aimed at creating a “neoileum” to treat bile acid malabsorption. To find optimal harvest sites for a dog model of stem cell transplantation, the exact location of peak IBAT expression in the donor ileum needs to be known. We therefore mapped IBAT function, IBAT mRNA, and IBAT protein in the ileum of Beagle dogs (N = 3). Mucosal samples were taken every 5 cm in the ileum and every 20 cm in the jejunum of each dog. Sodium-dependent (active) and sodium-independent (passive) taurocholate uptake rates were measured using a standardized everted sleeve technique. IBAT mRNA concentrations were determined by semiquantitative reverse transcriptase–polymerase chain reaction and IBAT protein concentrations by fluorometric immunohistochemical analysis. The small bowel measured 208±17 cm (mean±standard error of the mean). Active and passive uptake rates were found to follow distinct distribution curves. Significant active uptake was seen only at the terminal 50 cm and peaked at 479±176 pM/mm 2. Depending on location, active uptake accounted for approximately half of the total uptake. IBAT mRNA and protein distributions corroborated uptake curves. The terminal 10 to 50 cm of ileum has the highest bile acid uptake capacity. This short segment appears to be the most promising donor site for ileal stem cell transplants to create a “neoileum” in dogs.

Identification of a Ligand-binding Site in the Na+/Bile Acid Cotransporting Protein from Rabbit Ileum

Reabsorption of bile acids occurs in the terminal ileum by a Na(+)-dependent transport system composed of several subunits of the ileal bile acid transporter (IBAT) and the ileal lipid-binding protein. To identify the bile acid-binding site of the transporter protein IBAT, ileal brush border membrane vesicles from rabbit ileum were photoaffinity labeled with a radioactive 7-azi-derivative of cholyltaurine followed by enrichment of IBAT protein by preparative SDS gel electrophoresis. Enzymatic fragmentation with chymotrypsin yielded IBAT peptide fragments in the molecular range of 20.4-4 kDa. With epitope-specific antibodies generated against the C terminus a peptide of molecular mass of 6.6-7 kDa was identified as the smallest peptide fragment carrying both the C terminus and the covalently attached radiolabeled bile acid derivative. This clearly indicates that the ileal Na(+)/bile acid cotransporting protein IBAT contains a bile acid-binding site within the C-terminal 56-67 amino acids. Based on the seven-transmembrane domain model for IBAT, the bile acid-binding site is localized to a region containing the seventh transmembrane domain and the cytoplasmic C terminus. Alternatively, assuming the nine-transmembrane domain model, this bile acid-binding site is localized to the ninth transmembrane domain and the C terminus.

Intestinal absorption of bile acids: paradoxical behaviour of the 14 kDa ileal lipid-binding protein in differential photoaffinity labelling.

Photoaffinity labelling of brush border membrane vesicles from rabbit ileum with radiolabelled 3,3-azo and 7,7-azo derivatives of taurocholate identified integral membrane proteins of molecular masses 93 and 46 kDa, as well as a 14 kDa peripheral membrane protein, as components of the ileal Na+/bile acid transport system [Kramer, Girbig, Gutjahr, Kowalewski, Jouvenal, Müller, Tripier and Wess (1993) J. Biol. Chem. 268, 18035-18046]. Differential photoaffinity labelling in the presence of non-radiolabelled bile acid derivatives led, as expected, to a concentration-dependent decrease in the extent of labelling of the 93 and 46 kDa transmembrane proteins, which are the monomeric and dimeric forms of the ileal bile acid transporter protein. The extent of labelling of the 14 kDa ileal lipid-binding protein (ILBP), however, increased on the addition of unlabelled bile acids, the increase being dependent on the structure of the bile acid added. The possibility of artifacts was excluded by photoaffinity labelling experiments in the frozen state as well as by model calculations. The experimental results suggest that the binding of bile acids to ILBP can increase the affinity of ILBP for bile acids. These results would be in accordance with a substrate-load modification of transport activity and a positive-feedback regulation mechanism for active uptake of bile acid in the ileum.