FIGURE LEGENDS
FIGURE 1: Effect of 3 week tenapanor treatment on body weight, stool water content and stool pH in Cftr+/+ andCftr-/- mice: (a) Schematic description of experimental protocol. (b) Initial body weight of the male and female mice that did or did not develop obstructions. (c) The body weight remained stable during the 3 week gavage period in theCftr+/+ mice and (d) slightly decreased in theCftr-/- mice, irrespective of treatment with tenapanor or vehicle. (e) A significantly higher stool water content was seen in the tenapanor treated treated Cftr+/+and (f) Cftr-/- mice, in comparison to the vehicle treated groups. (g) Increased stool alkalinity 3 hours post gavage with tenapanor. Each dot represents one mouse.Cftr+/+ : n=10 in both tenapanor treated and vehicle treated groups; Cftr-/- : n=12 in the tenapanor treated and n=13 in the vehicle treated group. Data is represented as mean ± SEM. # is used to represent the statistics performed between the two different genotypes and * is used to represent the statistics performed between the vehicle and tenapanor groups. # and * p < 0.05.
FIGURE 2: Faster GI transit time (GTT) in Tenapanor treated mice: (a,c) GTT was measured once per week, and it decreased in bothCftr +/+ and Cftr -/-mice from week 1 to week 3, and was decreased after gavage with tenapanor compared to vehicle. (b,d) The longitudinal assessment of the stool water content during the 5 hours post gavage showed a significant increase with tenapanor compared to vehicle treatment. 0h indicates stool samples collected pre gavage. Each dot in the GI transit measurement represents one mouse, with the same mouse then being simultaneously assessed for stool water content. Data is represented as mean ± SEM. *p < 0.05.
FIGURE 3: Significantly lower incidence of obstructions in Tenapanor treated Cftr-/- mice: (a) Treatment with tenapanor resulted in a significant reduction of the incidence of intestinal obstructions in Cftr-/- mice compared to the vehicle treated Cftr-/- mice. (b) A representative image of the obstruction between ileum to proximal colon is shown. All mice but one were suspected to have suffered an obstruction while alive and were sacrificed prior to death. *p < 0.05.
FIGURE 4: Reduced cryptal hyperproliferation in Tenapanor treated Cftr-/- mice: (a) Representative HE images of jejunum, ileum, proximal colon and mid-distal colon inCftr+/+ and Cftr-/-mice treated either with vehicle or tenapanor. (b) Crypt-villus length or crypt depth measured showed reduced cryptal-villus prolongation in ileum and crypt prolongation in proximal colon of in the tenapanor treated Cftr-/- mice. (c) Muscle layer thickness displayed an increase in ileum, proximal colon and mid-distal colon of Cftr-/- mice, with no difference between vehicle and tenapanor treated group. Each dot represents one mouse. For each mouse, 5 different (not serial) sections per segment were analysed and averaged. Veh – vehicle, Ten – tenapanor, UO – unobstructed, O – obstructed. Data is represented as mean ± SEM. # is used to represent the statistics performed between the two different genotypes and * is used to represent the statistics performed between the vehicle and tenapanor groups. # and * p < 0.05.
FIGURE 5: Decrease in mucus accumulation in Tenapanor treatedCftr-/- mice: (a) Representative images of Muc2 immunohistochemistry (major component of the secreted colonic mucin) of jejunum, ileum, proximal colon and mid-distal colon inCftr+/+ and Cftr-/-mice treated with either vehicle or tenapanor. (b) Fluorescent intensity (I.D) ratio of Muc2 in relation to Dapi immunofluorescence (c) localized to the goblet cell thecae (intracellular mucus pool) and d) localized to the luminal compartment (secreted mucus). (e) mRNA expression ofMuc1 in these intestinal segments. Each dot represents one mouse. Veh – vehicle, Ten – tenapanor, UO – unobstructed, O – obstructed, I.D – Integrated density. Data is represented as mean ± SEM. # is used to represent the statistics performed between the two different genotypes and * is used to represent the statistics performed between the vehicle and tenapanor groups. # and * p < 0.05.
FIGURE 6: Reduced numbers of mast cells in in Tenapanor treatedCftr-/- mice: (a) Representative images of toluidine blue stained mast cells (indicated by black arrows) in the jejunum, ileum, proximal colon and mid-distal colon inCftr+/+ and Cftr-/-mice treated with either the vehicle or tenapanor. (b) Quantified mast cell counts within the selected intestinal segments. Each dot represents one mouse. Veh – vehicle, Ten – tenapanor, UO – unobstructed, O – obstructed. Data is represented as mean ± SEM. # is used to represent the statistics performed between the two different genotypes and * is used to represent the statistics performed between the vehicle and tenapanor groups. # and * p < 0.05.
FIGURE 7: Neutrophil infiltration in obstructedCftr-/- mice treated with vehicle: (a) Representative images of MPO stained neutrophils (indicated by white arrows) in the jejunum, ileum, proximal colon and mid-distal colon inCftr+/+ and Cftr-/-mice treated with either the vehicle or tenapanor (n=5/genotype/group/intestinal segment). (b) Counting of MPO positive cells show an obvious neutrophils infiltration in the ileum and proximal colon of obstructed Cftr-/- mice treated with vehicle. (c) Ly6g mRNA expression further confirmed this observation. Each dot represents one mouse. Veh – vehicle, Ten – tenapanor, UO – unobstructed, O – obstructed. Data is represented as mean ± SEM. # is used to represent the statistics performed between the two different genotypes and * is used to represent the statistics performed between the vehicle and tenapanor groups. # and * p < 0.05.
FIGURE 8: Inflammation in obstructedCftr-/- mice treated with vehicle:The mRNA expression of pro-inflammatory cytokines (a) Tnfα , (b)Mcp1 and (c) Il1β are not significantly different between genotypes or between the tenapanaor vs vehicle treated mice without an obstruction, but show a dramatic increase in the ileum and proximal colon of obstructed Cftr-/- mice. Each dot represents one mouse. Veh – vehicle, Ten – tenapanor, UO – unobstructed, O – obstructed. Data is represented as mean ± SEM. # is used to represent the statistics performed between the two different genotypes and * is used to represent the statistics performed between the vehicle and tenapanor groups. # and * p < 0.05.