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.