The Biliary Bicarbonate Umbrella in Fibrosing Cholangiopathies

Cholestatic liver diseases account for 10% of liver transplantations in Europe
(1). Unfortunately, the etiology and pathogenesis of fibrosing cholangiopathies
such as primary sclerosing cholangitis, primary biliary cholangitis and
IgG4-related hepatobiliary disease remains enigmatic. Recently, we introduced
the hypothesis of the *biliary HCO3- umbrella*. This hypothesis states that
cholangiocytes create a protective apical environment by secreting HCO3- and
that this *biliary HCO3- umbrella* is stabilized by an apical cholangiocyte
glycocalyx. The apical cholangiocyte glycocalyx is a 20- to 40-nm
membrane-bound barrier which is composed of glycoproteins that (1) could
possibly form a trap for secreted HCO3- and (2) repulse bile acids away from
the cholangiocyte cell surface. The resulting alkaline apical environment
created by HCO3- secretion and its retention in the apical glycocalyx would be
able to keep toxic glycine-conjugated bile acids in their deprotonated, polar
and membrane-impermeant state. We reasoned that impairment of the HCO3-
secretion machinery or disruption of the stabilizing glycocalyx would result in
protonation of glycine-conjugated bile acids, rendering them apolar and capable
of crossing the membrane where they could lead to detrimental damage in
cholangiocytes and lead to chronic fibrosing cholangiopathies such as PSC
Importantly, we were able to experimentally confirm parts our hypothesis in
vitro and have concluded from our findings that defects in the *biliary HCO3-
umbrella* may play a critical role in the development of fibrosing
cholangiopathies: Impairment of HCO3- secretion and degradation of the
glycocalyx resulted in increased bile acid permeation and damage to
cholangiocytes, others have recently confirmed these findings. RNA sequencing
performed on cholangioids derived from primary sclerosing cholangitis patients
demonstrated that 39 genes were significantly altered compared to non-primary
sclerosing cholangitis controls, 6 of these genes are implicated in glycocalyx
formation.
Knowing that disruption of biliary HCO3- umbrella renders cholangiocytes
vulnerable to bile acid permeation and to cholangiocyte damage raises the
following pivotal questions:
(1) Is the structure, composition and barrier function of the biliary HCO3-
umbrella impaired in fibrosing cholangiopathy patients?
(2) Is it possible to stabilize the biliary HCO3- umbrella, thereby preventing
bile acid permeation and cholangiocyte damage, and possibly discovering a new
therapeutic target for fibrosing cholangiopathies?

Based on our previous work we put forward two hypotheses:
(1) The biliary HCO3- umbrella is disrupted on a structural, compositional
and/or functional level in fibrosing cholangiopathies, thereby losing its
protective barrier function against toxic bile acids, and thus playing an
important role in the pathogenesis of fibrosing cholangiopathies.
(2) The biliary HCO3- umbrella can by stabilized through pharmacological
interventions, thereby preventing bile acid permeation and subsequent
cholangiocyte damage.

Although previous experiments support our hypothesis, this knowledge has mostly
been gained in cell models that are by now outdated/inferior. Additionally, the
previous cell models did not allow us to culture cholangiocytes from subjects
with fibrosing cholangiopathies. In recent years new cell culturing techniques
have become available that make it possible to culture so called cholangioids
(*bile ducts in a dish*). This culturing technique allows us to directly
culture cholangiocytes from fibrosing cholangiopathy subjects and control
subjects in vitro, thereby enabling us to greatly advance our understanding of
fibrosing cholangiopathies, better test our hypotheses, and possibly open up
new avenues for therapy.

Primary Objective:
Assess if the structure, composition and/or barrier function of the biliary
HCO3- umbrella is intrinsically altered in cholangioids and liver biopsies
derived from fibrosing cholangiopathy subjects compared to non-fibrosing
cholangiopathy subjects and if this alteration leads to increased bile acid
permeation and cholangiocyte damage.

Secondary Objectives:
Compare the effects of potentially therapeutic agents such as UDCA conjugates,
norUDCA, FXR agonists and fibrates on the structure, composition and barrier
function of biliary HCO3- umbrella in order to protect cholangioids against the
malicious effects of toxic bile acids.

Firstly, treating physicians will ask possible eligible subjects if they have
interest in participating in the study. They will be asked if the investigator
can contact them and inspect their clinical file (EPIC) to see if subjects are
eligible.

Secondly, eligible subjects will be informed about the study and asked for
their informed consent.

Thirdly, when informed consent is provided subjects will undergo their already
planned elective interventions.

Fourthly, subject material will be collected from these interventions.

Fifthly, subject material will be used for laboratory studies. Subject material
from liver tissue biopsies will be used for TEM and confocal microscopy.
Whereas subject material collected with ERCP, PTC, Whipple procedures and liver
resections will be used to isolate stem cells to derive cholangioids from these
stem cells. Blood and duodenal fluid aspirate will be used to correlate
experimental findings to clinical parameters.

Control group: The control group has to consist of subjects undergoing the same
interventions as we need excess to their subject material. The diagnoses for
the control group are determined on the basis that we expect their condition
not to affect the biliary HCO3- umbrella and thereby forming an appropriate
control group for our experimental studies.
Duration: Subjects will be included over a four year time period. For the
extended use and storage of subject material a biobank application will be
submitted.
Setting: The collection of subject material will take place in Amsterdam UMC
location AMC. Subject material will be processed and experiments performed at
the Tytgat Institute for Liver and Intestinal Research. Subjects will be
followed up via their treating physicians.

The only additional procedure that subjects will undergo is bile aspiration
during ERCP.

The ERCP endoscope will be equipped with a 5-French catheter that is inserted
through an existing channel in the ERCP endoscope. After entry to a
non-strictured region of the midportion of the common bile duct is obtained,
gentle suction will be applied to the catheter and approximately 2-5mL of bile
will be collected in a sterile tube. This tube will be transported on ice to
the laboratory, where we start with the in vitro derivation of cholangioids
from bile.

In case the physician that performs the ERCP judges that bile aspiration would
take prolong the procedure to the disadvantage of the patient, no bile
aspiration will be performed.

Nature and extent of the burden: All subjects will have to go through the
process of informed consent, in order to agree that their tissue and/or bile
will be used for scientific research. In addition, subjects will be asked to
give informed consent regarding access to their clinical information, which is
needed to correlate experimental data to clinical conditions.

Risks: Based on the literature we assess that there are no additional risks
attached to bile and duodenal fluid aspiration during ERCP. Although aspiration
of bile and duodenal fluid during ERCP is not standard practice, it is used for
diagnostic purposes and it is a relatively quick and simple procedure. Most
importantly, bile and duodenal fluid aspiration during ERCP have shown to be
safe, which will be discussed in section 13. Blood collection, as known, is a
procedure with minimal additional risks.

Benefits: There will be no direct benefits for participating subjects.
The justification for the proposed study is that we expect indirect long-term
future benefits for the whole group of fibrosing cholangopathy subjects, which
would result from a better understanding of the etiology and pathogenesis of
fibrosing cholangiopathies. This could hopefully translate into better
therapeutic targets for fibrosing cholangiopathies.

Although the current available literature demonstrates that there is no
increased risk of bile or duodenal fluid aspiration during ERCP in various
patient groups, we will take the following measures to reduce the potential
risks:
- The exclusion of vulnerable subjects (ICU admitted, clinically unstable,
pregnant).
- The judgment of the performing endoscopist: if bile or duodenal fluid
aspiration will disadvantageous to the subject (for e.g. due to an already
difficult or lengthy ERCP procedure); no bile aspiration will be performed.

With the starting point that the current literature does not demonstrate and
additional risks and our measures to prevent subjects from being at risk we
believe it would be acceptable for subjects to participate in the study.