Tenapanor treatment results in decreased mucus accumulation incftr-/- intestine
Immunostaining against Muc2, the major component in intestinal secreted mucus, showed that tenapanor treatment reduced the total Muc2 mediated fluorescence intensity in relation to the Dapi mediated fluorescence intensity, compared to vehicle treatment (Figure 5a,b). In order to determine whether the decrease was due to intracellular Muc2 in the goblet cell thecae, or in the luminal (mucosa-attached plus secreted mucus), we optically separated the epithelium from the lumen in each section, and quantified the two components (Supplementary. Figure 1a). Because of the many different parameters that had to be studied in the intestine of the mice, including the mucosa adherent microbiome (the results of which will be published by the group of Soraya Shirazy-Beechey), it was technically not possible to perform a Carnoy fixation that preserves the firmly adherent mucus layer. Therefore the quantification of the extracellular mucus in the luminal compartment is only approximate. Nevertheless the data show a significantly increased accumulation of mucus in the Cftr -/- mice (Figure 5a-c). Tenapanor significantly reduced the mucus accumulation in the ileum and proximal colon of the cftr -/-mice compared to vehicle treatment, while it did not affect these parameters in cftr+/+ intestine (Figure 5a-d). In the segments proximal to the site of obstruction (ileum and proximal colon), mucus accumulation in the intracellular and luminal compartment was prominent, but the two components could often not be distinguished with certainty. Muc2 mRNA expression was not different in the small intestine in either genotype or in the two treatment groups, but it was non-significantly reduced compared to Cftr+/+in the colon, similar to previous observations (Supplementary Figure 1b) (Kini et al. 2020). In contrast, mRNA expression of the membrane-resident Muc1 was increased in theCftr -/- ileum and proximal colon, and tenapanor treatment reduced Muc1 expression relative to vehicle treatment (Figure 5e).