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Comparison of intestinal expression of the apical sodium‐dependent bile acid transporter between dogs with and without chronic inflammatory enteropathy

Background Intestinal absorption of bile acids is mediated by the apical sodium‐dependent bile acid transporter (ASBT). Fecal bile acid dysmetabolism has been reported in dogs with chronic inflammatory enteropathy (CIE). Objective Characterization of ASBT distribution along the intestinal tract of control dogs and comparison to dogs with CIE. Animals Twenty‐four dogs with CIE and 11 control dogs. Methods The ASBT mRNA and protein expression were assessed using RNA in situ hybridization and immunohistochemistry, respectively. The concentrations of fecal bile acids were measured by gas chromatography‐mass spectrometry. The fecal microbiota dysbiosis index was assessed with a quantitative polymerase chain reaction panel. Results In control dogs, ASBT mRNA expression was observed in enterocytes in all analyzed intestinal segments, with highest expression in the ileum. The ASBT protein expression was restricted to enterocytes in the ileum, cecum, and colon. Dogs with CIE had significantly decreased expression of ASBT protein in the ileum (P = .001), which was negatively correlated with histopathological score (ρ = −0.40; P corr = .049). Additionally, dogs with CIE had a significantly increased percentage of primary bile acids in feces compared to controls (P = .04). The fecal dysbiosis index was significantly higher in dogs with CIE than in control dogs (P = .01). Conclusions and Clinical Importance These findings indicate that ileal protein expression of ASBT is downregulated in dogs with CIE. This change may be linked to the inflammatory process, intestinal dysbiosis, and fecal bile acid dysmetabolism observed in these patients.

CIE can be classified as food-responsive, antibiotic-responsive, or steroid-responsive. 2,3 Bile acid (BA) dysmetabolism, characterized by an increased proportion of primary BAs in the feces, has been described in human patients with inflammatory bowel disease (IBD) 4,5 and in dogs with CIE. [6][7][8] Bile acid malabsorption in people and rodents can cause diarrhea because of increased colonic secretion of water and electrolytes, increased intestinal permeability, and impaired lipid digestion. 9 Primary BAs, namely cholic acid (CA) and chenodeoxycholic acid (CDCA), are synthesized from cholesterol by hepatocytes, and subsequently conjugated with glycine or taurine before excretion in bile into the small intestinal lumen. 10 Approximately 90% of conjugated BAs are actively reabsorbed in the ileum through the apical sodiumdependent bile acid transporter (ASBT) and returned to the liver via the portal system. 10 Bile acids can be deconjugated by the gut microbiota in the distal small intestine and large intestine. 10 After deconjugation, the microbiota can 7α-dehydroxylate CA and CDCA to form the secondary BAs deoxycholic acid (DCA) and lithocholic acid (LCA), respectively. 10 Bile salt hydrolases responsible for BA deconjugation are produced primarily by Gram-positive commensal bacteria. 11 The generation of secondary BA by 7α-dehydroxylation is restricted to a narrow phylogenetic group of commensal bacteria 11 within Clostridium clusters XIVa and VI 11 and Eubacterium species. 12 In both humans and dogs, IBD and CIE are associated with intestinal dysbiosis, 13,14 which may cause impaired BA metabolism because of defective biotransformation. 5 The ileum is considered the main site of BA uptake in many mammalian species 15 and the ASBT, expressed primarily in this site, is considered the major pathway for intestinal uptake of conjugated primary BAs in rodents and humans. 10 The ASBT can be found in lower abundance in other portions of the small and large intestine, 16 as well as on cholangiocytes in the liver 17 and on the epithelium of the proximal renal tubules. 18 Physiologically, ASBT transcription is downregulated by activation of the nuclear BA receptor in enterocytes, known as farnesoid X receptor, by BAs in the intestinal lumen. 8 Studies in people with IBD have identified decreased expression of ASBT [19][20][21] because of repression of gene expression by inflammatory cytokine signaling. 22 Our objectives were to characterize the distribution of ASBT expression along the canine gastrointestinal tract and to compare ASBT mRNA and protein expression between dogs with CIE and control dogs. Additionally, associations among ASBT expression with the canine inflammatory bowel disease activity index (CIBDAI), histopathological scores, fecal BAs, and fecal dysbiosis index (DI) were investigated. cumulative score ranges from 0 to 18). 23 Serum albumin concentration was determined in all dogs with CIE and serum cobalamin concentration was measured in 11/24. None of the CIE dogs had received immunomodulatory drugs or UDCA in the month before sample collection. Most of the CIE dogs (21/24) had not received antibiotics in the month before sample collection. Three dogs had received antibiotics within 7 days of sample collection; these individuals were not included in the portion of the study that evaluated the fecal microbiota and concentrations of fecal BAs. Endoscopic (20/24) or full-thickness (4/24) biopsy samples were obtained from the ileum and colon of all dogs with CIE. Histological changes were classified according to the World Small Animal Veterinary Association Gastrointestinal Standardization Group histopathologic criteria using a grading system (0 = normal, 1 = mild lesions, 2 = moderate lesions, and 3 = severe lesions). 24 A cumulative histopathological score calculated as the sum of individual lesions scores was assigned to the ileum (0-30) and colon (0-24) for each CIE dog. Fecal samples stored at −80 C were available for 11/24 CIE dogs and were used to measure fecal BA concentrations and to determine the DI.

| Immunohistochemistry and image analysis
Immunohistochemistry was performed on samples of ileum and colon from 24 dogs with CIE and 11 control dogs. The FFPE samples were cut at 3 μm, adhered to charged slides, and deparaffinized. Heatinduced antigen retrieval was performed, followed by blocking of endogenous peroxidases. Sections were incubated with a goat antimouse solute carrier family 10, member 2 (SLC10A2) polyclonal antibody (#PA5-18990; Thermo Fisher Scientific, Rockford, Illinois) diluted 1:300 for 1 hour. Sections were incubated with goat immunoglobulin G (IgG) in lieu of the primary antibody as negative controls.
After incubation, slides were washed and then incubated with a mouse monoclonal anti-goat IgG-biotin secondary antibody (#sc-2023; Santa Cruz Biotechnology, Dallas, Texas). Slides then were incubated with avidin and biotinylated horseradish peroxidase. The target antigen was revealed by incubation in peroxidase substrate and 3,3 0diaminobenzidine chromogen. Slides were counterstained with Mayer's hematoxylin and were mounted using a xylene-based medium.
Ten random fields in regions of well-oriented villi were captured at ×400 magnification per slide with a digital camera for bright field microscopy (DP73; Olympus, Tokyo, Japan) by using cellSens standard software (Olympus, Tokyo, Japan). Images were analyzed by an immunohistochemistry image analysis toolbox for ImageJ software. 25 The area corresponding to the immunolabeling was automatically evaluated by the plug-in and the number of pixels corresponding to the labeled area was recorded for each image. The mean number of pixels corresponding to the immunolabeled area was calculated for the ileum and colon of each case.

| In situ hybridization and scoring
For specific detection of ASBT mRNA, RNA ISH using 20 probes targeting region 89-1032 of canine SLC10A2 mRNA (NM_001002968.1) was used. The ISH was performed on samples of ileum and colon from 24 dogs with CIE and 11 control dogs. The FFPE samples were cut at 3 μm, mounted on charged slides, and the RNAscope 2.5 HD red assay was performed according to the manufacturer's protocol (RNAscope; Advanced Cell Diagnostics, Hayward, California). Consecutive sections were incubated with a positive control probe targeting canine RNA polymerase II subunit A to verify RNA quality and a nonspecific bacterial RNA (dapB gene) probe was used as a negative control probe. Chromogenic detection with fast red was performed using alkaline phosphatase-based detection. The final deposits were red dots or clusters, with each dot corresponding to an mRNA copy. 26 Ten random fields in areas of villi were captured at ×400 magnification for each slide with a digital camera (DP73; Olympus) for bright field microscopy by using cellSens standard software (Olympus). The number of dots/cell and clusters was manually counted. The labeling was categorized into 6 scores: (0) negative, no staining or < 1 dot per 10 cells; (1) minimal, 1-3 dots/cell; (2) mild, 4-10 dots/cell with up to 10% of the dots forming clusters, (3) moderate, 10-15 dots/cell with 10%-20% of the dots forming clusters, (4) marked, 15-20 dots/cells with 20%-30% of the dots forming clusters, and (5) diffuse, with >20 dots/cell and >30% of the dots forming clusters. The mean score was calculated for the ileum and colon of each dog.

| Fecal bile acids
The concentrations of fecal BAs were measured in 11 dogs with CIE with available feces and 11 control dogs, utilizing methods established by Guard. 8  weights. Bile acid data were reported in μg/mg of lyophilized fecal content in addition to being expressed as a percent of total BAs measured. The deconjugated primary BAs CA and CDCA were combined to represent total primary BAs measured, and LCA, DCA, and UDCA were combined to represent total secondary BAs.

| Fecal dysbiosis index
The fecal DI was assessed in 11 dogs with CIE with available feces and 11 control dogs. The DNA was extracted from samples of 100 mg of feces using the MoBio Power soil DNA isolation kit (QIAGEN Inc., Germantown, Maryland) according to the manufacturer's instructions.
A quantitative polymerase chain reaction (qPCR) panel consisting of 8 bacterial groups: total bacteria, Faecalibacterium, Turicibacter, Escherichia coli, Streptococcus, Blautia, Fusobacterium, and Clostridium hiranonis was performed as previously described. 28 The qPCR data were expressed as the log amount of DNA (fg) for each particular bacterial group/10 ng of isolated total DNA. Results were imported into a mathematical algorithm for the calculation of a single numerical value, the DI. A negative DI indicates normobiosis, whereas a positive number indicates dysbiosis. 28

| Statistical analyses
The datasets were tested for normality and equality of variances using

| ASBT mRNA and protein expression in the intestinal tract of control dogs
On immunohistochemistry, ASBT protein was identified in the ileum, cecum, and colon. The duodenum and jejunum were negative. The ileum had the highest levels of ASBT immunolabeling, followed by the colon and cecum. Within the cellular compartment, immunolabeling was located within the apical membrane of enterocytes. In the ileum ( Figure 1A), the labeling was continuous and homogeneous in enterocytes of the villi, but absent in the crypts. In the colon (Figure 2A) and cecum, the labeling was multifocal and restricted to superficial enterocytes.
On ISH, ASBT mRNA expression was distributed in enterocytes throughout the villi in the small intestine, in the mucosa of the large intestine, and in the crypts of all intestinal segments. Expression of ASBT mRNA was minimal in the duodenum and jejunum, marked to diffuse in the ileum ( Figure 1C), and mild to moderate in the cecum and colon ( Figure 2C). Within the cell compartment, ASBT mRNA was located both in the nucleus and cytoplasm of enterocytes.

| ASBT mRNA and protein expression in the ileum and colon of CIE dogs
The location and distribution of the immunolabeling and mRNA expression in CIE dogs were the same as described in the control dogs. However, CIE dogs had significantly decreased immunolabeling for ASBT protein in the ileum (median, 206 pixels; range, 0-17 809 pixels; P < .001; Figures 1B and 3A) compared to control dogs (median, 6191 pixels; range, 800-21 955 pixels; Figure 1A). At mRNA levels, ASBT expression in the ileum of CIE dogs (Figures 1D and 3B) was numerically lower (median score, 3.21; range, 0.8-4.85), but not significantly different from control dogs (median score, 4.35; range, 3-4.95; P = .06; Figure 1C).

| Fecal dysbiosis index
Dogs with CIE had a higher fecal DI (mean AE SD, 1.77 AE 4.46, P = .01) than did the control dogs (mean AE SD, −2.17 AE 1.7; Figure 5). However, the individual log values for each bacterial group included in the DI were not significantly different between the 2 groups (Table 1) Table S1).

| DISCUSSION
Our study indicated that the ileum is the intestinal segment with the highest expression of ASBT mRNA and protein in dogs. Dogs with CIE had significantly decreased protein levels of ASBT in the ileum when The ASBT distribution in the control dogs was similar to that described in humans 16 and rodents. 22,29 As in other species, the ileum appears to be the main intestinal segment responsible for BA uptake FIGURE 3 Comparison of ASBT protein and mRNA expression in the ileum and colon between control dogs and dogs with CIE. In the ileum, the median immunolabeled area for ASBT protein (A) was significantly decreased in dogs with CIE (P < .001) when compared to control dogs. ASBT mRNA expression in the ileum (B) was not significantly higher in control dogs than in CIE dogs (P = .06). In the colon, ASBT protein (C) (P = .45) and mRNA (D) (P = .85) expression was similar for dogs with CIE and controls. Bars represent the median. *Significantly different but also may reflect cell damage and loss of transporters. The inflammatory process is considered a major mechanism for clinical signs in human patients with IBD and dogs with CIE. 1 However, it has been hypothesized that poor BA absorption because of ASBT inhibition may directly contribute to diarrhea in people with IBD. 9 Lower ASBT expression is reported primarily in human patients with ileal inflammation, but also can be observed in patients with inflammation limited to the colon. 21,35 The method of sample collection (ie, endoscopic or fullthickness biopsies) did not interfere with the analysis of ASBT expression because the distribution of ASBT mRNA and protein was limited to epithelial cells within the mucosa. Although a positive correlation was observed between mRNA expression and protein expression of ASBT in the ileum, the difference in mRNA levels between dogs with CIE and control dogs did not reach significance. This finding could be explained by post-transcriptional modifications of mRNA 36 or could reflect protein loss associated with epithelial injury secondary to inflammation. 37 Because the dogs with CIE had decreased ASBT protein expression in the ileum, one might expect BA malabsorption with increased loss of fecal BAs, as described in people with irritable bowel syndrome 38 or Crohn's disease. 39 However, in our study, the total concentration of fecal BAs in CIE dogs was similar to that of the controls. In humans, various methods have been used to diagnose BA malabsorption, including measurement of fecal BA concentrations, determination of retention of labeled BA analogs such as selenium homotaurocholic acid, and measurement of plasma concentrations of metabolites from BA synthesis such as lathosterol, 7a-hydroxycholesterol, or 7a-hydroxycholest-4-en-3one (C4). 40,41 To date, only 1 report has addressed this issue in dogs with CIE, in which serum C4 concentrations suggested BA malabsorption in 3/17 dogs. 42 Although our study did not identify overt BA malabsorption in dogs with CIE, the increased percentage of primary BAs in feces does suggest BA dysmetabolism. This also was identified in dogs with CIE in another recent study. 8 Similar to our findings, increased proportions of CDCA and decreased proportions of DCA have been reported in fecal samples of human patients with irritable bowel syndrome. 43 The increased proportion of primary BAs could be explained by a compensatory increase in de novo synthesis of BAs by hepatocytes 43 or by decreased bacterial biotransformation. 11 The intestinal microbiota is the sole metabolic pathway for BA transformation. 43 Thus, intestinal dysbiosis with a decrease in bacteria with bile salt hydroxylase activity can lead to decreased deconjugation and dehydroxylation of BAs. 11 Although the individual values for the bacterial taxa analyzed in this study were not significantly different, the DI was a reliable indicator of dysbiosis in the dogs with CIE. The abundance of a single taxon may not consistently distinguish between health and disease, 28 and for this reason the DI is preferred. The positive correlation between the abundance of C. hiranonis and the percentage of secondary BAs in feces suggests that this bacterium might play an important role in BA dehydroxylation in dogs. 28,44 In a previous study, C. hiranonis was found to be decreased in the feces of dogs with CIE. 28 Because dogs with CIE have decreased ASBT protein levels in the ileum, therapies that reestablish ASBT expression might be beneficial. 21 Corticosteroids are used routinely in the treatment of humans with IBD and dogs with CIE 10,45 and have been demonstrated to restore ASBT expression in people, 21 either by decreasing the levels of inflammatory cytokines that repress ASBT expression or by direct induction of ASBT transcription via activation of glucocorticoid receptors. 10 Further studies are needed to better characterize ASBT expression in different types of CIE in dogs (ie, steroid-responsive, food-responsive, and antibiotic- FIGURE 5 The composition of the fecal microbiota is represented as a dysbiosis index (DI). The median DI for dogs with CIE was significantly higher than for controls (P = .01). Bars represent the mean. *Significantly different