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\begin{document}

\title{The Na\textsuperscript{+}/H\textsuperscript{+} exchanger NHE3 inhibitor
tenapanor prevents intestinal obstructions in
\textsuperscript{}CFTR-deleted mice}



\author[1]{Xingjie Tan}%
\author[2]{Archana Kini}%
\author[1]{Dorothee Roemermann}%
\author[1]{Ursula Seidler}%
\affil[1]{Medizinische Hochschule Hannover}%
\affil[2]{Hannover Medical School}%


\vspace{-1em}



  
  \date{June 16, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background and purpose: Mutations in the CFTR chloride channel result in
intestinal o-structive episodes in cystic fibrosis (CF) patients and in
CF animal models. This study explores the possibility of reducing the
frequency of obstructive episodes in the
\emph{Cftr}\textsuperscript{-/-} mice by the oral application of a gut
selective NHE3 inhibitor tenapanor and searches for the underlying
mechanisms involved. Experimental approach: Sex and age-matched
\emph{Cftr}\textsuperscript{+/+} and \emph{Cftr}\textsuperscript{-/-}
mice were orally gavaged twice daily with 30mgkg\textsuperscript{-1}
tenapanor or vehicle for a period of 21 days. Body weight and stool
water content was assessed daily and gastrointestinal transit time (GTT)
once weekly. The mice were sacrificed when an intestinal obstruction was
suspected or after 21 days, and stool and tissues were collected for
further analysis. Key results: 21 day tenapanor application resulted in
a significant increase in stool water content, stool alkalinity, and a
significant decrease in GTT in \emph{Cftr}\textsuperscript{+/+} and
\emph{Cftr}\textsuperscript{-/-} mice. Tenapanor significantly reduced
obstructive episodes to 8\% compared to 46\% in vehicle treated
\emph{Cftr}\textsuperscript{-/-} mice and prevented mucosal
inflammation. A decrease in cryptal hyperproliferation, mucus
accumulation and mucosal mast cell number was also observed in tenapanor
compared to vehicle treated unobstructed
\emph{Cftr}\textsuperscript{-/-} mice. Conclusion and implications: Oral
tenapanor application prevented obstructive episodes in CFTR deficient
mice and was safe in \emph{Cftr}\textsuperscript{+/+} and
\emph{Cftr}\textsuperscript{-/-} mice. These results suggest that
tenapanor may be a safe and affordable adjunctive therapy in cystic
fibrosis patients to alleviate constipation and prevent recurrent DIOS.%
\end{abstract}%



\sloppy


\textbf{The NHE3 inhibitor tenapanor prevents intestinal obstructions in
CFTR-deleted mice}

Xinjie Tan, Archana Kini, Dorothee R\selectlanguage{ngerman}ömermann, Ursula Seidler

Department of Gastroenterology, Hannover Medical School, Hannover,
Germany

Running title: Tenapanor prevents intestinal obstruction in CF mice

Author for correspondence:

Ursula Seidler, Department of Gastroenterology, Hepatology and
Endocrinology, Hannover Medical School, Carl Neuberg Straße 1, D 30625
Germany, Tel.: +495115329427; Fax No. +49 5115328428, email:
Seidler.Ursula@mh-hannover.de

\emph{Word count:} 3848

\emph{Acknowledgements:} We acknowledge the help of all members of the
institute for animal research of the MHH. A part of the graphical
abstract was adapted from ``Administration Techniques for Mice - Part
3'', by BioRender.com (2022). Retrieved from
https://app.biorender.com/biorender-templates.

\emph{Conflict of interests:} The authors have no conflict of interest
to declare.

\emph{Funding:} Cystic Fibrosis Trust SRC 011 and the Deutsche
Forschungsgemeinschaft grants Se460/19-1 and For5046 Se460/22-1.

\emph{Authorship contributions:} X.T., A.K and U.S designed the study,
X.T., A.K and D.R. performed experiments, X.T., A.K and U.S. wrote the
manuscript.

\emph{Data availability:} The original data are available from the
authors upon reasonable request.

Bullet points:

\emph{What is already known} :

NHE3 inhibitor tenapanor increases luminal fluidity and alkalinity
in\emph{Cftr} \textsuperscript{-/-} mice during singlepass intestinal
perfusion studies.

\emph{What this study adds:}

3 week oral tenapanor application normalized intestinal transit time,
reduced obstructive episodes, and increased survival.

It also reversed crypt-villus elongation, reduced mucus accumulation and
prevented intestinal inflammation.

\emph{Clinical significance:}

Tenapanor maybe a safe and affordable adjunctive therapy in cystic
fibrosis patients to alleviate constipation and prevent recurrent DIOS

\textbf{Abstract}

\textbf{Background and purpose} : Mutations in the CFTR chloride channel
results in intestinal obstructive episodes in cystic fibrosis (CF)
patients and in CF animal models. This study explores the possibility of
reducing the frequency of obstructive episodes in the\emph{Cftr}
\textsuperscript{-/-} mice by the oral application of a gut selective
NHE3 inhibitor tenapanor and searches for the underlying mechanisms
involved.

\textbf{Experimental approach:} Sex and age-matched\emph{Cftr}
\textsuperscript{+/+} and \emph{Cftr} \textsuperscript{-/-}mice were
orally gavaged twice daily with 30mg kg\textsuperscript{-1}tenapanor or
vehicle for a period of 21 days. Body weight and stool water content was
assessed daily and gastrointestinal transit time (GTT) once weekly. The
mice were sacrificed when an intestinal obstruction was suspected or
after 21 days, and stool and tissues were collected for further
analysis.

\textbf{Key results:} 21 day tenapanor application resulted in a
significant increase in stool water content, stool alkalinity, and a
significant decrease in GTT in \emph{Cftr} \textsuperscript{+/+}
and\emph{Cftr} \textsuperscript{-/-} mice. Tenapanor significantly
reduced obstructive episodes to 8\% compared to 46\% in vehicle
treated\emph{Cftr} \textsuperscript{-/-} mice and prevented mucosal
inflammation. A decrease in cryptal hyperproliferation, mucus
accumulation and mucosal mast cell number was also observed in tenapanor
compared to vehicle treated unobstructed\emph{Cftr}
\textsuperscript{-/-} mice.

\textbf{Conclusion and implications:} Oral tenapanor application
prevented obstructive episodes in CFTR deficient mice and was safe
in\emph{Cftr} \textsuperscript{+/+} and \emph{Cftr}
\textsuperscript{-/-}mice. These results suggest that tenapanor may be a
safe and affordable adjunctive therapy in cystic fibrosis patients to
alleviate constipation and prevent recurrent DIOS.

\textbf{Key words:} mucoviscidosis, DIOS, mucus, intestinal fluid
absorption, sodium hydrogen exchange, chloride channel



\textbf{Introduction}

Cystic fibrosis (CF) patients develop intestinal obstructions, called
``meconium ileus'' in newborn and ``distal intestinal obstructive
syndrome'' (DIOS) in juveniles and adults. Radiologic signs and clinical
symptoms are that of mechanical ileus. Surgery is to be avoided, if at
all possible, and conservative treatment consists of orally and rectally
applied Gastrografin (meglumine diatrizoate), polyethylene glycol (PEG)
lavage (20\%), and oral laxatives and enemas (Houwen et al., 2010).
Nevertheless, the need for surgery is high, the incidence of obstruction
related segmental colectomies is increasing, with high morbidity and
mortality (Hite et al., 2022; Sharma, Morton, Peckham \& Jayne, 2012).
Recent Cochrane analyses revealed a clear lack of evidence for
preventive as well as therapeutic treatment strategies, and suggested
that randomised controlled trials need to be conducted (Carroll, Green
\& Gilchrist, 2021; Gilchrist, Green \& Carroll, 2021).

A recent survey revealed a high rate of abdominal complaints with a
likely origin in the lower gut in CF patients not suffering from
recurrent DIOS episodes (Hayee et al., 2019). Other studies demonstrated
a correlation between abdominal symptoms and an increase of inflammatory
markers in the stool (Beaufils et al., 2020; Jaudszus et al., 2022),
undermining the hypothesis derived from CF animal models that
inflammation, secondary to bacterial overgrowth, predisposes to
obstructive episodes, disease severity, and abdominal complications (De
Lisle, 2007; Dorsey \& Gonska, 2017; Talebi et al., 2022). However, this
hypothesis has not been validated in animal models or CF patients.

It has also been shown that the treatment with CF modulators and
correctors reduces CF-associated inflammation (Tétard et al., 2020).
Nevertheless, many of the patients having undergone surgery for DIOS
were already on CF corrector therapy, which is also not available for
all CF mutations. This suggests that additional intestine targeted
therapies that improve the sequelae of CFTR dysfunction, namely the
reduced gut fluidity, high luminal acidity, mucus hyper-viscosity,
increased transit time, microbial dysbiosis and mucosal inflammation and
thereby hopefully reduce obstructive episodes in the intestine, are
urgently needed. Because it is obviously difficult to perform
double-blind placebo controlled studies in this highly compromised group
of patients, it is important to carefully test pharmacological
strategies to alleviate CF related intestinal disease in suitable animal
models.

All currently available CF animal models display a very high incidence
of intestinal obstruction, leading to death unless prevented by
surgical, genetic or pharmacological rescue (Borowitz \& Gelfond, 2013;
Clarke, Gawenis, Franklin \& Harline, 1996; Rogers et al., 2008; Stoltz
et al., 2013). \emph{Cftr} \textsuperscript{-/-} mice do not display
pancreatic or pulmonary disease, but consistently develop intestinal
obstructive episodes. Hence, we considered this model ideal to test
intestine-targeted drug therapies that have been approved for the
treatment of constipation-prone irritable bowel disease and are believed
to increase epithelial fluid secretion, decrease fluid absorption, or
both.

In a previous report, we had examined the efficacy of the guanylate
cyclase 2C ligand linaclotide, prostaglandin E1 analogue lubiprostone,
and intestine-specific
Na\textsuperscript{+}/H\textsuperscript{+}exchanger isoform 3 (NHE3)
inhibitor tenapanor on jejunal and colonic fluid balance and alkaline
output in anesthetized both\emph{Cftr} \textsuperscript{-/-} mice and in
F508del mutant mice\emph{in vivo} , titrating each drug to the minimal
concentration that elicited a significant increase in gut fluidity and
alkalinity (Tan et al., 2021). Although each of the drugs was able to
increase jejunal alkaline output and reduce jejunal fluid absorption,
linaclotide had no effect on the colon, and lubiprostone needed to be
applied in much higher concentrations than were likely present in the
gut lumen with the approved dosages for humans, suggesting that the
anti-constipation effect may be due to an effect on motility rather than
fluid balance. Tenapanor was able to decrease fluid absorption and
increase alkaline output with similar efficacy in \emph{Cftr}
\textsuperscript{-/-} and\emph{Cftr} \textsuperscript{+/+} mice, and did
so in concentrations that were reasonably close to what can be expected
in the gut lumen with the approved dosage for humans. We therefore chose
tenapanor to test its efficacy to prevent obstructive episodes
in\emph{Cftr} \textsuperscript{-/-} mice when given by oral gavage for
21 days. We also aimed to explore the effect of tenapanor on the
hallmarks of CF intestinal dysfunction, namely the reduced gut fluidity
and alkalinity, mucus accumulation, cryptal hyperproliferation and
villus elongation, slowed gastrointestinal transit time, and mucosal
inflammation.



\textbf{MATERIALS AND METHODS}

\textbf{Animals and experimental protocol}

All experiments were performed with wild type mice
(\emph{Cftr\textsuperscript{+/+}} ) and CFTR null mice
(\emph{Cftr\textsuperscript{-/-}} ) (FVB/N-CFTRtm1CAM) (Ratcliff et al.,
1993). Both\emph{Cftr\textsuperscript{+/+}} and
\emph{Cftr\textsuperscript{-/-}}mice were bred and maintained at
Hannover Medical School as previously described (Xiao et al., 2012),
except for the use of an energy-rich diet for transgene animals
(1414MOD141003, Altromin, Lage, Germany) instead the low-fiber diet
(C1013, Altromin, Lage, Germany), after the mice had survived the first
2 months of age. \emph{Cftr \textsuperscript{-/-}}and their respective
wild type littermates were co-housed (unless the males need to be
separated due to fighting), and received identical diet (1414MOD141003,
Altromin, Lage, Germany) and the osmotic laxative drinking solution
(Oralav, 40 mmol
l\textsuperscript{-1}Na\textsubscript{2}SO\textsubscript{4}, 75 mmol
l\textsuperscript{-1}NaHCO\textsubscript{3}, 10 mmol
l\textsuperscript{-1} NaCl, 10 KCl, 23 g l\selectlanguage{english}\textsuperscript{-1} PEG 4000)
to prevent obstruction and enhance survival post weaning.

The experimental protocol was performed in age-matched mice (9-13 weeks)
with similar percentage of males and females. Post withdrawal of
Oralav,\emph{Cftr \textsuperscript{+/+}} and
\emph{Cftr\textsuperscript{-/-}}mice were intragastrically gavaged twice
daily (with a gap of 10-12 hours) with either the vehicle (PBS, pH 6.0)
or with Tenapanor (30mg kg\textsuperscript{-1}) for 21 days. Mice were
assigned into the vehicle or Tenapanor treated group randomly. The mice
were monitored daily and sacrificed either at the end of the experiment
or if obstruction was suspected. Mice were sacrificed when a suspicion
of obstruction was present over two consecutive observation periods (1
observation period every 12 hours, lasting 12 hours). Suspicion of
obstruction was present when the mice had a prominent abdomen, a hunched
body composure, no passage of feces during one 12 hour observation
period, or a score of 3 or higher in the mouse wellbeing score. The
severity and scoring was assessed based on a published severity
assessment system which is recommended by our institute for animal
research (Bleich \& Tolba, 2017). The abdomen was opened, the intestine
was dissected, photographed and the site of obstruction was verified.
Post sacrifice, the intestinal tissues were harvested for RT-PCR and
histology.

The experimental protocol was approved by the Hannover Medical School
Committee and an independent committee assembled by the local
government. Animal studies are reported in accordance with the ARRIVE
guidelines 2.0 and the guidelines from the British Journal of
Pharmacology (Percie du Sert et al., 2020). The application and
permission numbers are Az. 33.14-42502-04-14/1549 and Az.
33.12-42502-04-19/3197 for breeding ``stressed strains'' and Az.
33.9-42502-04-18/2829 for experimental procedures.

\textbf{Whole GI transit time}

The total GI transit time (GTT) using Carmine red stained food pellets
was measured once per week as previously described with a few
modifications (Kini et al., 2022). Briefly, the mice were fasted
overnight (approximately 8 hours) and were placed in a clean transparent
empty cage prior to the first gavage of the day. They were then fed the
red pellets. The total GI transit time was measured as the interval
between the first food intake until the appearance of the first red
stool pellet.

\textbf{Stool water content}

Stool water content was measured every morning prior to gavage as
previously described (Kini et al., 2020). Stool pellets were also
collected every hour for 5 hours during the GI transit time measurement.

\textbf{Stool pH measurement}

Luminal stool pH was measured in \emph{Cftr} \textsuperscript{+/+}
and\emph{Cftr} \textsuperscript{-/-} mice pre and 3 hours post gavage
with Tenapanor. One to two fresh stool pellets were collected, placed in
50\selectlanguage{greek}μ\selectlanguage{english}l unbuffered distilled water and the pH determined with a pH-strip
(Carl Roth, Karlsruhe, Germany). Following this, the pellets were
homogenized and the pH was measured via a pre-calibrated TitraLab TIM
865 pH meter (Hach, D\selectlanguage{ngerman}üsseldorf, Germany) with a Sension+ 5209 pH
electrode (Hach, Düsseldorf, Germany).

\textbf{Histology and immunohistochemistry}

Dissected intestinal segments (jejunum, ileum,colon) were fixed in 4\%
paraformaldehyde and embedded in paraffin according to standard
procedures. 3\selectlanguage{greek}µ\selectlanguage{english}m thick sections were stained with Hematoxylin and Eosin
(HE) and basic histological parameters such as crypt-villus length,
crypt depth and muscle layer thickness were assessed (Alshamy,
Richardson, H\selectlanguage{ngerman}ünigen, Hafez, Plendl \& Al Masri, 2018). Crypt-villus
length was defined as the distance from the crypt base to the apex of
the villus in the jejunum and ileum. Crypt depth was measured from the
basement of crypt to the surface epithelium in the proximal and
mid-distal colon. Three to eight villi or crypts were measured and
averaged for every section. Muscle layer thickness was measured from
serosa to inner muscularis mucosae. Intestinal sections were also
stained with Toluidine blue to detect mast cells numbers (Norkina, Kaur,
Ziemer \& De Lisle, 2004). Images were acquired under the Leica DFC295
light microscope. Mast cells were then counted in five different
sections per segment. The results are expressed as cells per
mm\textsuperscript{2} (image field area).

The dissected intestinal segments were processed for immunohistochemical
(IHC) staining as previously described (Kini et al., 2020). The sections
were incubated overnight at 4°C with primary antibodies to Muc2 (1:200,
Santa Cruz Biotechnology, Heidelberg, Germany, Cat\#sc-15334, RRID:
AB\_2146667) and MPO (1:250, Proteintech, Planegg-Matinsried, Germany,
Cat\# 22225-1-AP, RRID:AB\_2879037). It was then incubated with the
corresponding secondary antibody (Goat anti-rabbit Alexa 568; 1:500,
Thermofisher, Waltham, MA, USA, Cat \#A-11036, RRID AB\_10563566) and
DAPI to detect the nuclei. The slides were mounted with Mowiol 4-88 and
images were acquired on the Olympus FluoView\selectlanguage{english} FV1000 confocal
microscope. Muc2 was quantified as per previously described with a few
modifications (Honda et al., 2017). Briefly, the intensity of green
fluorescence indicating MUC2 stained mucins were quantified using Image
J software. A region of interest (ROI) was determined (Supplementary
Figure 1a) individually for intracellular and secreted mucins. Followed
by normalizing the detected area with the background, the integrated
density (I.D) ratio of Muc2 to Dapi was calculated for total, as well as
for the intracellular and secreted mucins. The additive Muc2 to Dapi
ratio of the intracellular and secreted mucins represents the total
Muc2. MPO positive cells were counted by eye in image taken at 40X
magnification in five different sections per segment. The results are
expressed as cells per mm\textsuperscript{2} (image field area). .

\textbf{Quantitative PCR}

Isolation of RNA, cDNA conversion and quantitative real-time PCR from
the various intestinal segments (jejunum, ileum, proximal colon and
mid-distal colon) were done as per manufacturer's instructions and as
previously described (Tan et al., 2021). Ribosomal protein S9 (RPS9) was
used as housekeeping gene. The list of primers used can be found in
Supplementary Table 1. The total RNA was extracted with the RNeasy(r) Mini
Kit (Qiagen GmbH, Hilden, Germany) based on the manufacturer's
instructions. 1\selectlanguage{greek}μ\selectlanguage{english}g RNA was reverse transcribed using the QuantiTect\selectlanguage{ngerman}®
Reverse Transcription Kit (Qiagen GmbH, Hilden, Germany). cDNA was then
diluted 1:20 with DNase free water, and 4\selectlanguage{greek}μ\selectlanguage{english}L of the dilution was used as
a template for PCR. Each reaction contained 5\selectlanguage{greek}μ\selectlanguage{english}L qPCRBIO SyGreen Mix
Lo-ROX (PCR Biosystems, Dueren, Germany), 4 \selectlanguage{greek}µ\selectlanguage{english}l template and an
appropriate amount of primers.

\textbf{Data and statistical analysis}

The study design included a priori power analysis to determine the
sample size for each experiment/subgroup. We used data from
prior/similar experiments to determine the effect sizes and SDs. These
were used in a power analysis with the software G*Power (Faul,
Erdfelder, Lang \& Buchner). To determine the necessary sample sizes, we
assumed a type I error of \selectlanguage{greek}α \selectlanguage{english}= 5\% and a statistical power of at least
80\%. Mice were allocated to the experimental groups by a different
person than the experimenter and this person also regenotyped the mice
after killing and allocated the genotype to the mouse number. A fully
blinded experiment is not possible, because the experienced experimenter
has a high chance of correctly guessing the genotype based on the
different phenotypes of knockout and wild-type mice. Sample sizes were
calculated as biological replicates and were allocated equally to the
experimental groups. Studies were designed to generate groups of equal
size; however, accidental deaths (spontaneous or euthanasia due to
disease) occurred during the preparatory period (transfer of the mice
after group assignment to an animal experiment laboratory, one week
obligatory acclimatization, removal of PEG laxative at day 1 of the
experimental period), resulting in slightly uneven numbers in
the\emph{Cftr\textsuperscript{-/-}} groups. The data were analyzed with
GraphPad Prism Version 8.0.2, adhering to the British Journal of
Pharmacology's recommendations for experimental design and analysis in
pharmacology (Curtis et al., 2018). All results are presented as mean \selectlanguage{ngerman}±
SEM. The body weight, stool water content and GI transit time are
averaged for each time point. Unpaired Students't-test (for parametric
data with normal distribution) or non-parametric Mann-Whitney U-test was
used for the comparisons between vehicle and tenapanor treated groups
and/or within genotype. Area under the curve was used to analyze the
daily body weight and stool water content. Obstruction and survival
curve was analysed by the Kaplan-Meier log-rank test. For multiple
comparisons between the genotypes, the one-way ANOVA (for parametric
data with normal distribution) was used with the Bonferroni post hoc
analysis only if the F value of the ANOVA reached significance. If the
normality test failed with one or both groups, the non-parametric
Kruskal-Wallis test was performed. Significant differences are indicated
as: \# and *p \textless{} 0.05.

\textbf{Materials}

Tenapanor was purchased from Adooq Bioscience (Irvine, CA, USA). Carmine
red was from SERVA Electrophoresis (Heidelberg, Germany). Mayer's
Hematoxylin Solution, Eosin Y Solution and Toluidine blue were all from
Sigma-Aldrich (Deisenhofen, Germany).

\textbf{RESULTS}

\textbf{Increased stool water content, more alkaline stool pH, but no
difference in body weight between vehicle and tenapanor treated mice.}

\emph{Cftr\textsuperscript{+/+}} and \emph{Cftr}
\textsuperscript{-/-}mice were intragastrically gavaged with either
vehicle (1X PBS, pH 6.0) or tenapanor (30mg kg\textsuperscript{-1})
twice a day for 21 days in the absence of oral PEG-containing laxative
(Figure 1a). Weight and stool water content was recorded daily. Body
weight was slightly higher in the \emph{Cftr\textsuperscript{+/+}} group
due to a higher number of males (Figure 1b). It was stable during the
treatment period during the 3week period in the
\emph{Cftr\textsuperscript{+/+}} group and slightly decreased in the
\emph{Cftr\textsuperscript{-/-}} group (possibly due to the stress
related to the gavage), but was not different between tenapanor- and
vehicle-treated mice in either group (Figure 1c,d).

A significantly higher stool water content was observed in the
tenapanor-treated \emph{Cftr\textsuperscript{+/+}}
and\emph{Cftr\textsuperscript{-/-}} throughout the gavage period (Figure
1e,f). The vehicle treated \emph{Cftr\textsuperscript{-/-}} mice
displayed a decrease of stool water content post removal of Oralav
(Figure 1f). This was less prominent in
the\emph{Cftr\textsuperscript{+/+}} mice (Figure1e). In a subset of
mice, we also measured the stool pH before and 3 hours after gavage with
tenapanor (Figure 1g), and found the pH to be significantly lower
in\emph{Cftr\textsuperscript{-/-}} compared
to\emph{Cftr\textsuperscript{+/+}} mice, and to be significantly
increased after tenapanor gavage.

\textbf{Accelerated GI transit time upon treatment with Tenapanor}

Gastrointestinal transit time (GTT) is prolonged in CF patients and in
CFTR deficient mice (De Lisle, 2007; Dellschaft et al., 2021; Malagelada
et al., 2020; Vitko et al., 2016). We therefore measured GTT after oral
gavage of 30 mgkg\textsuperscript{-1} tenapanor or vehicle once per
week. Compared to \emph{Cftr\textsuperscript{+/+}} ,
the\emph{Cftr\textsuperscript{-/-}} mice in the vehicle treated group
showed a significantly longer transit time (Figure 2a,c). Upon treatment
with Tenapanor, a significant decrease of transit time was observed from
week 1 onwards in the \emph{Cftr\textsuperscript{-/-}} mice and in week
3 in the \emph{Cftr\textsuperscript{+/+}} mice (Figure 2a,c). This
reduction of GTT in both vehicle treated and tenapanor
treated\emph{Cftr\textsuperscript{+/+}} and
\emph{Cftr\textsuperscript{-/-}}mice from week 1 to week 3 was
independent of the effect of tenapanor on the stool water content in the
5 hours during the determination of GTT which showed a significant
difference between vehicle and tenapanor treated mice of both genotypes
(Figure 2b,d). Interestingly, a difference in the pre-gavage (0h) stool
water content (not significant until week 2, but significant in week 3)
between the vehicle and tenapanor treated
\emph{Cftr\textsuperscript{+/+}} mice but not in
the\emph{Cftr\textsuperscript{-/-}} mice was observed (which would be a
time point of approx. 11 hours after their last gavage). This may
indicate a different durability of tenapanor action
in\emph{Cftr\textsuperscript{+/+}} and
\emph{Cftr\textsuperscript{-/-}}intestine.

\textbf{Significant reduction in obstructive episodes by Tenapanor}

Treatment with Tenapanor resulted in a significantly lowered number of
obstructive episodes in \emph{cftr} \textsuperscript{-/-} mice (8.3\%
vs. 46.2\%, p\textless{}0.05) (Figure 3a), and a significantly better
survival of 91.7\% with tenapanor compared to 53.9\% with vehicle
treatment (because all mice with suspected obstruction were sacrificed).
In the \emph{Cftr} \textsuperscript{+/+} group, survival was 100\% both
with tenapanor and with vehicle treatment. The site of obstruction was
between the proximal and mid colon in most cases, and the segments in
which the fecal material had accumulated was mostly restricted to the
regions of the mid-distal ileum and the proximal colon (Figure 3b),
because obstruction was suspected early. Table 1 shows the age and sex
distribution of the obstructed and
unobstructed\emph{Cftr\textsuperscript{-/-}} mice. While the total group
of mice before gavage had near identical age and sex distributions in
both the vehicle and the tenapanor treated groups, the mice that
suffered an obstruction had a male and young age predominance.

\textbf{Tenapanor treatment reverses cryptal hyperproliferation in the
ileum and proximal colon}

Assessment of the cryptal and villus length, the mucosal integrity and
the muscle layer thickness was performed in all mice of the treatment
groups. In the ileum, proximal and mid-distal colon, vehicle
treated\emph{Cftr\textsuperscript{-/-}} mice displayed crypt
villus/crypt elongation and increased muscle layer thickness compared to
the\emph{Cftr\textsuperscript{+/+}} mice in the ileum and colon, but not
the jejunum (Figure 4a-c). Treatment with tenapanor alleviated the
crypt-villus/crypt elongation in the unobstructed ileum and proximal
colon compared to the vehicle treated
\emph{Cftr\textsuperscript{-/-}}mice (Figure 4b, two middle panels). The
increase in muscle layer thickness, seen in all segments except the
jejunum in the\emph{Cftr\textsuperscript{-/-}} mice, was not
significantly affected by tenapanor treatment. The jejunum did not
display significant differences between the groups.

\textbf{Obstructed segments display distorted architecture and loss of
mucosal integrity}

The obstructed group of mice displayed a highly distorted epithelial
architecture in the ileum and proximal colon, which were the segments
that were regularly affected by the accumulation of food particles and
fecal bacteria rich material (Figure 4a). The mucosal barrier was
clearly destroyed and a clear separation between intra- and
extracellular mucus was not possible in many instances, and areas were
identified where the epithelium was sloughed off the lamina propria. All
these changes had occurred in the live mouse within a relatively short
time span and demonstrates the severe ulcerative behaviour of the
luminal content in conjunction with the altered physiological state of
the intestinal segment.

\textbf{Tenapanor treatment results in decreased mucus accumulation
in\emph{cftr}\textsuperscript{-/-} 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 \emph{Cftr} \textsuperscript{-/-} mice
(Figure 5a-c). Tenapanor significantly reduced the mucus accumulation in
the ileum and proximal colon of the \emph{cftr}
\textsuperscript{-/-}mice compared to vehicle treatment, while it did
not affect these parameters in \emph{cftr\textsuperscript{+/+}}
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 \emph{Cftr\textsuperscript{+/+}}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
the\emph{Cftr} \textsuperscript{-/-} ileum and proximal colon, and
tenapanor treatment reduced Muc1 expression relative to vehicle
treatment (Figure 5e).

\textbf{Mast cell number reduced in Tenapanor treated intestine}

An increase in innate immune cells, particularly mast cells, has been
shown in the small intestine of \emph{Cftr\textsuperscript{-/-}} mice
(Norkina, Kaur, Ziemer \& De Lisle, 2004). Importantly, a recent study
highlighted the mast cell-dependent immune function to a worse survival
in F50del mutant mice (Philp et al., 2018). We therefore studied mast
cell numbers and the effect of tenapanor treatment. A significant
increase of Toluidine Blue stained mast cells were seen in all examined
intestinal segments of the \emph{Cftr\textsuperscript{-/-}} compared
to\emph{Cftr\textsuperscript{+/+}} mice (Figure 6a, b). Treatment with
tenapanor resulted in a significant reduction in the number of mast
cells in the jejunum and mid-distal colon of\emph{Cftr}
\textsuperscript{-/-} mice, which were also the segments with the
highest mast cell numbers in the epithelium (outside of Peyer´s
patches). In other segments, while there was a similar trend, it did not
reach statistical significance. No differences in mast cell number were
seen between vehicle and tenapanor
treated\emph{Cftr\textsuperscript{+/+}} mice groups (Figure 6a,b).

\textbf{Neutrophil infiltration in the segments proximal to the site of
obstruction}

Myeloperoxidase (MPO) staining was performed to assess neutrophil
infiltration. The neutrophil count in the different segments
of\emph{Cftr\textsuperscript{+/+}} and \emph{Cftr\textsuperscript{-/-}}
intestine were very low, but there was a significant increase in the
ileum and mid-distal colon between \emph{Cftr\textsuperscript{+/+}}
and\emph{Cftr\textsuperscript{-/-}} mice, independent of treatment.
Neutrophil infiltration was strongly increased in the segments
immediately proximal to the site of obstructions, but not in the
mid-distal colon, which was the segment that was always distal to the
obstruction (Figure 7a,b). To verify this finding, we measured the mRNA
expression of Ly6g, a marker for differentiated neutrophils and
granulocytes (Figure 7c). \emph{Ly6g} expression was much higher in the
segments proximal to the obstruction.

\textbf{Strong expression of proinflammatory cytokine expression in the
intestinal segments proximal to the obstruction}

Proinflammatory cytokine expression is more sensitive to detect low
level inflammation than MPO staining. \selectlanguage{greek}\emph{Tnfα} , \selectlanguage{english}\emph{Mcp1} ,
and\selectlanguage{greek}\emph{Il1β} \selectlanguage{english}mRNA levels were assessed in the different intestinal
segments of all gavaged mice (Figure 8a-c). The expression levels were
low in \emph{Cftr\textsuperscript{+/+}}
and\emph{Cftr\textsuperscript{-/-}} intestine, irrespective of
treatment, except in the case of an obstruction having occurred.
Proinflammatory cytokine expression levels were significantly increased
in all segments proximal to the obstructed site, but not in the segment
distal to the obstructed site (mid-distal colon). However, the relative
increase was approx. 10 fold higher in the ileum and proximal colon, the
segments that were in contact with the fecal matter, than in the
jejunum, which is usually free of fecal content when the obstruction was
clinically detected and the mice sacrificed. Please observe the
different y-axis of the individual bargraphs.

\textbf{Discussion}

The primary goal of the study was to assess the potential of the oral
intestine-specific NHE3 inhibitor tenapanor to prevent intestinal
obstruction in CFTR null mice. Several major obstacles were noticed
during the study preparation and had to be addressed: Tenapanor quickly
precipitates from aqueous solutions at neutral or alkaline pH. Since
microencapsulated tenapanor for use in mice is not available, it had to
be applied by gavage, and dose finding studies were necessary. We aimed
at finding a tenapanor dose and application frequency that resulted in a
stable but mild increase in stool water, because diarrhea is quickly
lethal for mice (Barone et al., 2009). 30
mgkg\textsuperscript{-1}tenapanor, applied twice daily by gavage,
resulted in a mild increase in stool water, and it did not significantly
influence the body weight of the mice. Surprisingly, the required dose
was the same in\emph{Cftr} \textsuperscript{-/-} and \emph{Cftr}
\textsuperscript{-/-}mice, suggesting relative independence of the CFTR
mediated route of fluid secretion and the NHE3 mediated route of fluid
absorption. It is likely that the microencapsulation of tenapanor
available for use in humans will permit lower doses in the CF
population. An optimal dosing regimen to prevent obstructive episodes in
CF patients without causing diarrhea or other unwanted side effects
needs to be established.

The \emph{Cftr} \textsuperscript{-/-} mice experienced a mild loss of
body weight in both the tenapanor and vehicle group, which may be
related to an increased stress level due to the twice daily gavage. The
lack of effect of tenapanor on body weight is an important finding,
because it is known that NHE3 function is required for PEPT1-mediated
dipeptide absorption (Thwaites \& Anderson, 2007). It is in agreement
with a lack of reduction of PEPT1-mediated substrate absorption during
short term tenapanor ingestion in healthy volunteers (Johansson et al.,
2017). However, NHE3 has also been shown to be involved in the
absorption of amino acids (Anderson \& Thwaites, 2005) and
micronutrients (Shawki et al., 2016). Therefore, careful observation is
necessary during long term studies in CF patients.

Replacing the drinking fluid containing the osmotic laxative with tap
water may result in an initiation of an obstructive episode within the
first day (which becomes evident only several days later); we therefore
started the tenapanor gavage on the last day of the oral laxative
administration. It is well known that the incidence of obstructive
episodes (always lethal) in \emph{Cftr} \textsuperscript{-/-} mice is
very high during weaning (Snouwaert et al., 1992), and 90\%\emph{Cftr}
\textsuperscript{-/-} mice are dead by day 30 unless preventive measures
are taken (Clarke, Gawenis, Franklin \& Harline, 1996), and decreases in
adulthood. Previous studies that aimed at pharmacological prevention of
obstructive episodes in\emph{Cftr} \textsuperscript{-/-} mice have
therefore administered the pharmaceutical agent to mice aged 3-6 weeks
(Walker, Simpson, Levitt, Boyle \& Clarke, 2006), and 20 days,
respectively (Lord et al., 2018). For our study, the mice had to
tolerate twice daily gavage and preferably not loose body weight due to
this procedure; we therefore chose the age range of 9-13 weeks. This
explains why the percentage of obstructions is lower in our study
compared to the studies mentioned above.

The primary goal of our study was the questions whether intestinal
obstructive episodes can be prevented by tenapanor in a test group at
high risk for obstructions, which is the CFTR null mouse (no CFTR
protein present). Although our study was conducted in an age group in
which the mice have a decreased risk compared to younger age, the
results showed that the risk was still high (46\% within the observation
time) and was reduced to 8\% with tenapanor treatment.

Apart from obtaining a positive result of the study, we wanted to
understand how tenapanor conveyed this reduction in obstructive episodes
at a cellular and molecular level. As previously studied in intestinally
perfused mice, the direct effect of tenapanor will be a reduction of
NHE3-mediated sodium and fluid absorption and an increase in the luminal
alkalinity in both the small and large intestine (Tan et al., 2021),
which was also seen in the stool of the mice in this study (Figure 1g).
A normalization of the delayed GTT in the tenapanor-treated\emph{Cftr}
\textsuperscript{-/-} mice is likely another key effect protecting the
mice against obstructions.

Somewhat surprisingly, tenapanor treatment resulted in the reversal of
other features of the intestinal abnormalities in\emph{Cftr}
\textsuperscript{-/-} mice. Of particular interest to us was the finding
that tenapanor treatment reversed the cryptal hyperproliferation in the
ileum and proximal colon. A hyperproliferative response has been
observed previously in\emph{Cftr} \textsuperscript{-/-} small (Gallagher
\& Gottlieb, 2001) and large intestine (Tan et al., 2021). The
intracellular pH of\emph{Cftr} \textsuperscript{-/-} deficient
enterocytes is more alkaline than that of corresponding \emph{Cftr}
\textsuperscript{+/+} cells (Simpson, Gawenis, Walker, Boyle \& Clarke,
2005). This feature is preserved in the crypts including the stem cells
of intestinal organoids (Liu, Walker, Cook, Ootani \& Clarke, 2012) and
is causally related to the increased proliferative rate, which is
preserved in\emph{Cftr} \textsuperscript{-/-} intestinal organoids
(Strubberg et al., 2018). The presence of NHE3 has been reported both in
undifferentiated and differentiated intestinal organoids (Foulke-Abel et
al., 2016). Immunohistochemical staining of NHE3 overlaps with that of
CFTR in the villus region and cryptal mouth region (Jakab, Collaco \&
Ameen, 2011). Therefore, it is possible that chronic tenapanor
application reverses in part some of the pathological effects of the
high intracellular pH on proliferative activity. A similar observation
was made by Bradford\emph{et al} . in their studies of mice which were
homozygous negative for CFTR, for NHE3, or for both genes. While
both\emph{Cftr} \textsuperscript{-/-} and \emph{Nhe3}
\textsuperscript{-/-}mice displayed an increased cell proliferation in
duodenal crypts, this increase was significantly reduced in the crypts
of\emph{Cftr\textsuperscript{-/-}/Nhe3\textsuperscript{-/-}} mice
(Bradford, Sartor, Gawenis, Clarke \& Shull, 2009). Thus, long term
tenapanor treatment, if proven safe in CF patients, may even have to
potential to lower the risk for GI malignancy.

Another striking observation in tenapanor treated mice was a reduction
in intestinal mucus accumulation. We previously reported an increase in
the goblet cell counts, as well as enlarged goblet cell theca, in the
mid-distal colon of \emph{Cftr} \textsuperscript{-/-} mice that did not
display evidence of inflammation (Kini et al., 2020; Tan et al., 2021).
Because of the well documented crypt-villus hyperproliferation
in\emph{Cftr} \textsuperscript{-/-} intestine (Gallagher \& Gottlieb,
2001; Tan et al., 2021), we used a fluorometric method of quantifying
the Muc2 immunofluorescence in relation to that of the Dapi stained
nuclei in this project. The total as well as the extracellular Muc2/Dapi
immunofluorescence was significantly reduced in tenapanor vs
vehicle-treated mice in the ileum and proximal colon, while it was very
high when an obstruction was present.

We also noted a shift in the expression of the \emph{Muc} genes, with a
non-significantly lower expression of \emph{Muc2} mRNA and a
significantly higher \emph{Muc1} mRNA. The 21 day treatment with
tenapanor reduced \emph{Muc1} mRNA expression compared to vehicle
treatment. The somewhat lower \emph{Muc2} mRNA expression was also seen
in the present study, with no effect of tenapanor. It has been shown
that \emph{Muc1} deletion, although not a major component of intestinal
mucin, reduces the thickness of the firmly adherent mucus layer
(Malmberg et al., 2006). Muc1 deleted CF mice have reduced amounts of
intestinal mucus and a better survival compared to Muc1-expressing CF
mice (Parmley \& Gendler, 1998). It is therefore likely that the
observed tenapanor induced changes in Muc1 expression and mucus
accumulation play a major role in reducing obstructive episodes.

The intestinal tract of patients with cystic fibrosis display an
inflammatory phenotype in the absence of bacterial infection or enzyme
induced fibrosing colitis. Multiple explanations have been provided,
including bacterial overgrowth due to dysmotility, oxidative stress,
intracellular accumulation of mutated CFTR protein, and lack of
pancreatic and/or intestinal defensins (Bruzzese et al., 2004; Crites et
al., 2015; Galli et al., 2012; Lisowska, Mdry, Pogorzelski, Szyd\selectlanguage{polish}ł\selectlanguage{english}owski,
Radzikowski \& Walkowiak, 2010; Raia et al., 2000; Werlin et al., 2010).
In mice, chronic exposure to PEG containing laxative in the drinking
fluid both increased survival and reduced the inflammatory signature in
the intestine of these mice (Bradford, Sartor, Gawenis, Clarke \& Shull,
2009; Clarke, Gawenis, Franklin \& Harline, 1996). We had previously
reported no difference in proinflammatory cytokines in the different
segments of \emph{Cftr} \textsuperscript{-/-} and\emph{Cftr}
\textsuperscript{+/+} littermates while on the PEG containing laxative,
yet several features that had been correlated with an absent CFTR
function (in addition to the reduced anion and fluid secretory response)
were present: increased enterocyte pH\textsubscript{i}, an increased
mucus content in the goblet cell theca, crypt-villus elongation,
decreased surface pH with fluid hyper-absorption (Kini et al., 2020; Tan
et al., 2021). In this study, a discreet but significant increase in the
number of mast cells was observed in the lamina propria of the
intestinal tract of the vehicle treated\emph{Cftr} \textsuperscript{-/-}
mice compared to\emph{Cftr} \textsuperscript{+/+} mice, which was
completely or partially prevented by tenapanor treatment. A very
discreet increase in neutrophil infiltration was seen in \emph{Cftr}
\textsuperscript{-/-} ileum and mid-distal colon, not accompanied by an
increase in any of the proinflammatory cytokines. In contrast, a strong
increase in neutrophil infiltration and in the expression of
proinflammatory cytokines was only seen in mice with an obstruction, and
was restricted to the intestinal segments proximal to the obstruction
that were filled with fecal matter. These segments also exhibited severe
alterations in the mucosal architecture. Thus, the intestinal
inflammation was a sequelae of the obstruction, and not its cause.

A relative shortcoming in our study is that the application mode of the
drug is relatively invasive, and therefore the study duration is fairly
short. The number of mice permitted for the study by the animal
committee was sufficient to reach the primary endpoint, but was not
enough to determine significance for the reversal of every studied
CF-associated intestinal abnormalities by tenapanor treatment in every
intestinal segment. When we performed the GTT experiments, we noticed
that the stool water content was still elevated in the\emph{Cftr}
\textsuperscript{+/+} mice 11 hours after the last dosing, but not in
the \emph{Cftr} \textsuperscript{-/-} mice, suggesting that more
frequent dosing, not feasible with this form of application, may have
been optimal. Obviously, this problem will not exist in CF patient
trials. For a tenapanor trial in CF patients, it will be necessary to
define the patient population at high risk for intestinal complications,
and preferably test its prophylactic potential. Patients with meconium
ileus at birth and prior DIOS episodes, and lung or liver transplanted
patients are at particularly high risk, and CF diabetes, pancreatic
insufficiency and female gender also carry a higher risk (Houwen et al.,
2010; Lavie et al., 2015; Munck et al., 2016). While corrector and
potentiator therapy is beneficial for CFTR-dependent functional outcome
parameters (Graeber et al., 2022; Ooi et al., 2018), adjunctive therapy
specifically targeting the constipation and obstructive episodes appears
necessary in many CF patient subgroups.

Tenapanor treatment is considered safe for humans (Block et al., 2021;
Sinagra et al., 2020). It has not been tested in children so far, and
the longest observation time in a published trial is 52 weeks. Given the
good safety profile, clinical trials testing the effect of tenapanor on
the intestinal symptoms of CF patients seem warranted.

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\textbf{Table 1:}\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& \textbf{Gender} & \textbf{Gender} & \textbf{Gender} & \textbf{Gender}
& \textbf{Age (weeks)} & \textbf{Age (weeks)}\tabularnewline
& \textbf{Vehicle} & \textbf{Vehicle} & \textbf{Tenapanor} &
\textbf{Tenapanor} & \textbf{Vehicle} &
\textbf{Tenapanor}\tabularnewline
& \textbf{Male} & \textbf{Female} & \textbf{Male} & \textbf{Female} &
&\tabularnewline
\textbf{Unobstructed} & 1 & 6 & 4 & 7 & 11.7 \selectlanguage{ngerman}± 0.522 & 11.5 \selectlanguage{ngerman}±
0.366\tabularnewline
\textbf{Obstructed} & 4 & 2 & 1 & 0 & 10.8 \selectlanguage{ngerman}± 0.477 & 9.0\tabularnewline
\textbf{Total} & 5 & 8 & 5 & 7 & 11.3 \selectlanguage{ngerman}± 0.365 & 11.3 \selectlanguage{ngerman}±
0.392\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Table 1: Gender and age at the start of the experimental period of the
mice that developed and obstruction and that did not.

\textbf{FIGURE LEGENDS}

\textbf{FIGURE 1: Effect of 3 week tenapanor treatment on body weight,
stool water content and stool pH in \emph{Cftr\textsuperscript{+/+}}
and\emph{Cftr\textsuperscript{-/-}} 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
the\emph{Cftr\textsuperscript{+/+}} mice and (d) slightly decreased in
the\emph{Cftr\textsuperscript{-/-}} mice, irrespective of treatment with
tenapanor or vehicle. (e) A significantly higher stool water content was
seen in the tenapanor treated treated
\emph{Cftr\textsuperscript{+/+}}and (f) \emph{Cftr\textsuperscript{-/-}}
mice, in comparison to the vehicle treated groups. (g) Increased stool
alkalinity 3 hours post gavage with tenapanor. Each dot represents one
mouse.\emph{Cftr\textsuperscript{+/+}} : n=10 in both tenapanor treated
and vehicle treated groups; \emph{Cftr\textsuperscript{-/-}} : 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 \textless{} 0.05.

\textbf{FIGURE 2: Faster GI transit time (GTT) in Tenapanor treated
mice:} (a,c) GTT was measured once per week, and it decreased in
both\emph{Cftr} \textsuperscript{+/+} and \emph{Cftr}
\textsuperscript{-/-}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 \textless{} 0.05.

\textbf{FIGURE 3: Significantly lower incidence of obstructions in
Tenapanor treated \emph{Cftr\textsuperscript{-/-}} mice:} (a) Treatment
with tenapanor resulted in a significant reduction of the incidence of
intestinal obstructions in \emph{Cftr\textsuperscript{-/-}} mice
compared to the vehicle treated \emph{Cftr\textsuperscript{-/-}} 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
\textless{} 0.05.

\textbf{FIGURE 4: Reduced cryptal hyperproliferation in Tenapanor
treated \emph{Cftr\textsuperscript{-/-}} mice:} (a) Representative HE
images of jejunum, ileum, proximal colon and mid-distal colon
in\emph{Cftr\textsuperscript{+/+}} and
\emph{Cftr\textsuperscript{-/-}}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
\emph{Cftr\textsuperscript{-/-}} mice. (c) Muscle layer thickness
displayed an increase in ileum, proximal colon and mid-distal colon of
\emph{Cftr\textsuperscript{-/-}} 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 \textless{} 0.05.

\textbf{FIGURE 5: Decrease in mucus accumulation in Tenapanor
treated\emph{Cftr\textsuperscript{-/-}} mice:} (a) Representative images
of Muc2 immunohistochemistry (major component of the secreted colonic
mucin) of jejunum, ileum, proximal colon and mid-distal colon
in\emph{Cftr\textsuperscript{+/+}} and
\emph{Cftr\textsuperscript{-/-}}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 of\emph{Muc1} 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 \textless{} 0.05.

\textbf{FIGURE 6: Reduced numbers of mast cells in in Tenapanor
treated\emph{Cftr\textsuperscript{-/-}} mice:} (a) Representative images
of toluidine blue stained mast cells (indicated by black arrows) in the
jejunum, ileum, proximal colon and mid-distal colon
in\emph{Cftr\textsuperscript{+/+}} and
\emph{Cftr\textsuperscript{-/-}}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
\textless{} 0.05.

\textbf{FIGURE 7: Neutrophil infiltration in
obstructed\emph{Cftr\textsuperscript{-/-}} 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
in\emph{Cftr\textsuperscript{+/+}} and
\emph{Cftr\textsuperscript{-/-}}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 \emph{Cftr\textsuperscript{-/-}} mice
treated with vehicle. (c) \emph{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
\textless{} 0.05.

\textbf{FIGURE 8: Inflammation in
obstructed\emph{Cftr\textsuperscript{-/-}}} \textbf{mice treated with
vehicle:}The mRNA expression of pro-inflammatory cytokines (a)
\selectlanguage{greek}\emph{Tnfα} , \selectlanguage{english}(b)\emph{Mcp1} and (c) \selectlanguage{greek}\emph{Il1β} \selectlanguage{english}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 \emph{Cftr\textsuperscript{-/-}} mice.
Each dot represents one mouse. Veh -- vehicle, Ten -- tenapanor, UO --
unobstructed, O -- obstructed. Data is represented as mean \selectlanguage{ngerman}± 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 \textless{} 0.05.

\textbf{Hosted file}

\verb`Tanetal_Figures.pptx`  available at \url{https://authorea.com/users/484951/articles/570535-the-na-h-exchanger-nhe3-inhibitor-tenapanor-prevents-intestinal-obstructions-in-cftr-deleted-mice}

\selectlanguage{english}
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