AQP1 in the Gastrointestinal Tract of Mice: Expression Pattern and Impact of AQP1 Knockout on Colonic Function

Abstract

Aquaporin 1 (AQP1) is one of thirteen known mammalian aquaporins. Its main function is the transport of water across cell membranes. Lately, a role of AQP has been attributed to other physiological and pathological functions including cell migration and peripheral pain perception. AQP1 has been found in several parts of the enteric nervous system, e.g., in the rat ileum and in the ovine duodenum. Its function in the intestine appears to be multifaceted and is still not completely understood. The aim of the study was to analyze the distribution and localization of AQP1 in the entire intestinal tract of mice. AQP1 expression was correlated with the hypoxic expression profile of the various intestinal segments, intestinal wall thickness and edema, as well as other aspects of colon function including the ability of mice to concentrate stools and their microbiome composition. AQP1 was found in a specific pattern in the serosa, the mucosa, and the enteric nervous system throughout the gastrointestinal tract. The highest amount of AQP1 in the gastrointestinal tract was found in the small intestine. AQP1 expression correlated with the expression profiles of hypoxia-dependent proteins such as HIF-1α and PGK1. Loss of AQP1 through knockout of AQP1 in these mice led to a reduced amount of bacteroidetes and firmicutes but an increased amount of the rest of the phyla, especially deferribacteres, proteobacteria, and verrucomicrobia. Although AQP-KO mice retained gastrointestinal function, distinct changes regarding the anatomy of the intestinal wall including intestinal wall thickness and edema were observed. Loss of AQP1 might interfere with the ability of the mice to concentrate their stool and it is associated with a significantly different composition of the of the bacterial stool microbiome.

Keywords: Aquaporin 1; enteric nervous system; hypoxia; intestinal oxygen gradient; myenteric plexus; submucosal plexus.

Figure 1

AQP1 expression in different parts of the gastrointestinal tract and co-localization with ganglia of the enteric nervous system. Figure 1 shows a representative picture from every segment of the gastrointestinal tract of WT mice. Immunohistochemistry showed an increased overall expression of AQP1 (red staining) in the small intestine particularly in the jejunum and ileum (scale bar 100 µm). Next to the serosa and submucosal glands in the esophagus, a high AQP1 expression was found in the mucosa. Depending on the segment, AQP1 was seen more prominently in the submucosal or myenteric plexus. The immunofluorescence staining confirmed the expression of AQP1 (green) (scale bar 100 µm). The calretinin positive ganglion was surrounded by AQP1 positive fibers in the absence of S100B positive glial cells in the stomach sample (scale bar 100 µm). In the colon sample there was a co-localization of APQ1, calretinin, and S100B in the nervous structures of the myenteric plexus (scale bar 50 µm). Nuclei were stained by DAPI (blue).

Figure 1

AQP1 expression in different parts of the gastrointestinal tract and co-localization with ganglia of the enteric nervous system. Figure 1 shows a representative picture from every segment of the gastrointestinal tract of WT mice. Immunohistochemistry showed an increased overall expression of AQP1 (red staining) in the small intestine particularly in the jejunum and ileum (scale bar 100 µm). Next to the serosa and submucosal glands in the esophagus, a high AQP1 expression was found in the mucosa. Depending on the segment, AQP1 was seen more prominently in the submucosal or myenteric plexus. The immunofluorescence staining confirmed the expression of AQP1 (green) (scale bar 100 µm). The calretinin positive ganglion was surrounded by AQP1 positive fibers in the absence of S100B positive glial cells in the stomach sample (scale bar 100 µm). In the colon sample there was a co-localization of APQ1, calretinin, and S100B in the nervous structures of the myenteric plexus (scale bar 50 µm). Nuclei were stained by DAPI (blue).

Figure 2

AQP1 expression and relation to hypoxia-related factors. (A) Scoring of AQP1 immunohistochemical staining and overall expression of AQP1 determined by immunohistochemical staining through scoring (left graph) and by integrated density picture analysis (right graph) in WT mice. In both graphs the highest amount of AQP1 was found in the small intestine (jejunum, followed by ileum). Both methods showed a similar pattern of AQP1 distribution. (B) AQP1 mRNA expression. The relative gene expression confirmed the high expression of AQP1 protein in the jejunum and ileum on the mRNA level. (C) HIF-1α mRNA expression and (D) PGK1 mRNA expression. The mRNA expression of the hypoxia-dependent genes HIF-1α and PGK1 was shown in WT and AQP1-KO mice. The highest amounts of HIF-1α and PGK1 were found in the small intestine corresponding to the AQP1 expression. In all organs the loss of APQ1 led to an increase in HIF-1α and PGK1 in the AQP1-KO compared to the WT mice. The increase in HIF-1α was significant in the duodenum (p-value < 0.0001) and the stomach (p-value < 0.0002). PGK1 was increased in the AQP1-KO mice compared to WT mice except for the stomach and ileum. The increase in PGK1 in the AQP1-KO compared with the WT mice was significant in the duodenum and the jejunum (p-value < 0.0001 and <0.007, respectively).

Figure 3

WT versus AQP1 Knockout: Analysis of the intestinal wall. (A) Intestine: Anatomical stuctures. The intestinal wall is comprised, from outside to inside, of the serosa, the longitudinal muscular layer, the myenteric plexus, the circular muscular layer, the submucosa with the submucosal plexus, and the mucosa. The measurements of the intestinal wall thickness were taken according to the labelling in the figure. (B) Differences in colon structure, It already can be visually observed in the immunohistochemical staining (both 20× magnification), that there were obvious differences in the intestinal wall structure between AQP1-KO and WT mice. (C) Intestinal wall thickness. Depicted are the thickness measurements of the entire intestinal wall in WT and AQP1-KO mice. In the stomach, jejunum, and ileum the loss of AQP1 led to a significant increase in abdominal wall thickness (p < 0.0001, p < 0.00001, and p-value < 0.002, respectively), while, as seen in B above, it can be confirmed that the intestinal wall thickness decreased significantly in the colon of AQP1-KO compared to WT mice (p < 0.006). (D) Muscular layer/(E) mucosal layer in (D,E) the thickness of the muscular layer and the mucosal layer of the abdominal wall are depicted for all intestinal segments separately. In both mucosal and muscular layers, the trends were similar to the measurements of the entire wall. However, a major increase in the mucosal layer was noted in stomach, jejunum, and ileum (p > 0.001, p < 0.000001, and p < 0.002, respectively), while both the mucosal and muscular layer decreased in the colon of AQP1-KO compared to WT mice (p < 0.0007 and p < 0.007, respectively). (F) Wet-to-dry ratio. The wet-to-dry ratio was determined by calculating: (wet weight–dry weight)/dry weight. An increase in intramural water content was observed in all segments.

Figure 4

Stool analysis. (A) Dry weight of stool. While there were no significant differences in the dry weight of AQP1-KO (n = 4) and WT (n = 5) mice stools, it is notable that the standard deviation of AQP1-KO mice was much higher than that of WT mice (ns: not significant). (B) Heat release of stool. Isothermal microcalorimetry of pooled mouse stools revealed a reduced heat release of AQP1-KO stools compared to that of WT over time (first graph), resulting in a higher overall metabolic activity of WT stool during the observed time period (second graph). This is a first hint at a changed composition of microbiota in the AQP1-KO mice compared to the WT mice. (C) Bacterial alpha diversity. Alpha diversity in the AQP1-KO (n = 4) and WT (n = 5) group were measured using the Shannon and the Simpson indices, respectively. In both indices the groups were not significantly different. (D) Bacterial beta diversity. Bray–Curtis dissimilarities among the samples after the pairwise dissimilarities were transformed using non-metric multidimensional scaling (NMDS) in order to display the outcomes in two-dimensions. The differences between the two groups (WT and AQP1-KO) were assessed using non-parametric multivariate analysis of variance showing in a significant difference (p = 0.013). (E) Bacterial abundance. Relative abundance of the single phyla are shown from AQP1-KO (left) and WT (right) mice. The higher abundance of verrucomicrobia in the AQP1-KO compared to the WT was significant. Bacteroidetes and Firmicutes both have a slightly lower abundance in the AQP1-KO. (ns = not significant).

Figure 5

Summary of findings and context.