THE BILIARY TREE AND CHOLESTASIS

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THE BILIARY TREE AND CHOLESTASIS

• Richard Hinton
• School of Biological Sciences
• University of Surrey

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Cholestasis and Jaundice

• Cholestasis means cessation of bile flow
• Jaundice means yellow staining of tissues
  especially the whites of the eyes in humans and
  the ears in rats and mice normally due to
  excessive amounts of bilirubin
• Cholestasis normally results in jaundice.
• Jaundice does not necessarily mean there is
  cholestasis

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Types of Jaundice

• Prehepatic The rate of formation of bilirubin
  greater
• than the capacity for removal. Normally
  associated with hemolysis.
• Hepatic a) Damage to liver cells stop
  conjugation and/or excretion. Associated
  with severe hepatocellular damage
• b) Failure to form bile
• c) Reflux of bile between hepatocytes
• Posthepatic a) Damage to intrahepatic bile
  ducts
• b) Obstruction of intra or, more usually
  extrahepatic bile ducts

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Sporadic Drug-induced Cholestasis In Humans

• A considerable number of newly-developed drug
  cause cholestasis in a small proportion of
  patients.
• This condition cannot be reproduced in
  experimental animals.
• Effects can be quite marked but are generally
  reversible
• In some cases it seems likely that an
  immunological mechanism is involved, in other
  cases it is clear it is not.
• The result may be withdrawal of the license
  resulting in enormous financial loss.

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Diagnosis Of Cholestasis

• 1) Jaundice associated with an increase in
  conjugated bilirubin. (In prehepatic jaundice or
  liver damage jaundice is associated with
  unconjugated bilirubin)
• 2) Raised serum alkaline phosphatase and
  5'-nucleotidase. In man gamma-glutamyl
  transpeptidase is a useful marker but generally
  oversensitive, in rats secretory IgA is the most
  sensitive marked available
• 3) Loss of ability to clear drugs normally
  excreted in the bile.
• NB There are no distinctive pathological changes
  common to all types of cholestasis.

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The biliary tree a reminder

• Bile is formed by secretion into bile canaliculi.
  These are small channels between hepatocytes
  which are delineated by tight junctions which
  prevent material discharged into the bile from
  leaking back between the hepatocutes. The
  centrilobular end of bile canaliculi is blind
• Enlargement of the bile canaliculi is limited by
  a corset of actin microfilaments. This forces
  the newly made bile to flow down the bile
  canaliculi towards the periportal zone.
• The hepatocytes of the extreme periportal zone
  make contact with bile duct lining cells and
  there is a very short stretch, called the canal
  of Hering, where bile flows in channels lined by
  a mixture of bile duct lining cells and
  hepatocyes

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Continued

• While each branch of the portal tree generally
  contains one portal venule and one hepatic
  arteriole there may be many bile ductules of
  varying size which fom an anatomising network.
• As the bile passes down the bile ducts there is
  resorbtion of water and low molecular weight
  nutrients such as sugars.
• In the majority of species (but not in rats) most
  of the time bile enters a small sack, called the
  gall bladder where there is further resorbtion by
  a mechanism similar to that which occurs in the
  loop of Henle, namely secretion of osmolytes into
  the interstitual fluid increase the tonocity
  attracting water across the tight junctions.
• Discharge of bile into the intestine is governed
  by the sphincter of Oddi. The precise anatomy
  here, especially the relation to the pancreatic
  ducts varies from species to species

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Composition of Bile

• The principle components of human bile are-
• 1) Bile salts (67). These are strong detergents
  whose role is to emulsify fat in the intestine.
• 2) Phospholipids (22)
• 3) Cholesterol (4)
• 4) Proteins (4.5)
• 5) Bilirubin and other conjugates lt2
• In addition bile contains the normal inorganic
  components of extracellular fluids and is
  enriched in bicarbonate, giving it a slightly
  alkaline pH.

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Formation of Bile

• Bile flow is closely related to the concentration
  of bile acids and salts in the blood. There is
  evidence for a small, bile salt independent flow
  but this is very small compared with the bile
  salt dependent flow. Transporting bile salts
  from the serum to the blood involves transport
  proteins on both the sinusoidal and the bile
  canalicular surfaces of the hepatocyte.
• To make matters worse the momenclature used for
  these proteins is being changed radically. I
  will follow the nomenclature used by Arrese and
  Trauner (Trends Mol.Med 9 (2003) 558-564 which is
  the principal source for this part of the lecture

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Transporters involved in biliary excreetion
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Uptake of bile acids and Salts

• Entry of the bile salts into the hepatocytes is
  largely mediated two carriers, one an "organic
  acid"/sodium co-transporters (NTCPs). This moves
  bile salts up their concentration gradient (no
  ATP hydrolysis is involved) but the laws of
  thermodynamics are preserved as sodium is moved
  down its concentration gradient. NB This process
  on the concentration of sodium in the cell
  remaining low. This is due to the action of the
  ATP-powered Na/K exchange carrier. Toxins such
  as ouabain which inhibit this cause cholestasis.
• The second group of systems, the organic anion
  transport proteins (OATPs) are sodium independent
  and multi-specific. Again they appear to be
  co-transporters moving GSH and bicarbonate ions
  out of the cell

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Movement of bile salts into the canaliculi

• The final transport into bile are pumps which
  utilize the energy from ATP to move bile salts
  and other candidates for biliary excretion
  against a large concentration gradient. These
  transport proteins were identified because their
  expression in tumours results in multiple drug
  resistance. Several families of these were found
  to have a common structure and they have now been
  gathered together as ABC proteins. These
  proteins are characterized by possessing 12
  intramembrane domains and two highly conserved
  ATP binding sites. ABCB11 (aka BSEPbile duct
  exchange pump) is the most important in moving
  bile salts. More on the others later.

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Transfer of water and plasma components

• Water and low molecular weight neutral componemts
  of plasma enter bile by flowing between cells
  (the paracellular pathway)..

The main osmotic attractants are the bile acid
and bile salts which are responsible for the
bile acid -dependent bile flow. The relatively
small bile acid independent flow is thought to
depend mainly on the excretion of glutathione.
Both these processes require "sieving" across the
tight junctions so that although the low
molecular weight components of plasma enter bile
readily, plasma proteins only pass in small
quantities and the rate of passage is inversely
related to size
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Continued

• Phospholipid, cholesterol and "enzymes"
• The detergent action of bile extracts
  phospholipids and some cholesterol from the
  plasma membranes of the hepatocytes lining the
  bile canaliculus. Some plasma membrane enzymes
  are extracted at the same time. Replenishment of
  the phospholipid seems to be by the action of
  MDR3 (ABCB4)
• Bilirubin glucuronides and other conjugates
• MDR1 (ABCB1) mediates the excretion of
  hydrophobic, mostly cationic, metabolites. MDR2
  (ABCC2) mediates the excretion of anionic
  conjugates including bilirubin and dyes. Yet
  others (eg ABCG5 and G8) transport steroids

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Entry by Transcytosis

• Material can also be moved from the sinusoidal to
  the bile canalicular face of the cell by
  transcytosis which is, for example, known to be
  the mechanism for transporting Immuno-globulin A
  (IgA) from plasma to bilein certain species
  Probably its most important role is to move
  plasma membrane proteins from the sinusoidal to
  the bile canalicular face of the cell. As shown
  in the diagram the process involves

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Or in words

• 1) Endocytosis at the sinusoidal surface of the
  cell of proteins to be transferred (including
  IgA-IgA receptor complexes.
• 2) Transfer to the endosome compartment where
  proteins are sorted into streams directed to
  lysosomes, back to the sinusoidal surface or to
  the bile canalicular surface of the cell
• 3) Discharge of the contents of the vesicles
  into the bile canaliculus.
• In addition to this immediate transcytosis
  about 5 of lysosomes are discharged into the
  bile each day

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Modification of Bile After Secretion

• 1) Some water and nutrients are resorbed from
  the bile duct into the blood vessels which form
  the peribiliary plexus
• 2) In some species the bile duct lining cells
  secrete a bicarbonate-rich fluid under the
  control of the hormone secretin.
• 3) In species which possess a gall bladder there
  is marked concentration of the bile by a
  mechanism which resembles counter-current
  resorbtion in the loop of Henle.

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Development of Cholestasis

• This this may veassociated with
• 1) Failure to secrete bile acids and bile salts
  - these are the principal water attractants and
  in their absence bile will not flow
• 2) Obstruction of the intrahepatic bile ducts,
  usually due to inflammation (cholangitis). It
  has been suggested that some cases bile may
  reflux through tight junctions between
  hepatocytes.

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Possible examples

• D-RING METABOLITES OF OESTROGENS
• Action immediate and reversible
• Clear-cut dose response
• Compete with taurocholate for binding site on
  plasma membrane
• Probably perturbs bile acid metabolism

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Important but puzzling

• CHOLESTASIS INDUCED BY OESTROGENS
• Oestrogen-associated cholestasis was noted after
  introduction of both oral contraceptives and
  anabolic steroids. Effects are observed about
  12h after administration
• The effects can be reproduced in mice and rats
• The effect appears to be on hepatocytes
• 1) Bile canaliculi are dilated and show bile
  plugs
• 2) BSP and bilirubin retention has been observed
• 3) With ethinyl estradiol there are changes in
  the permeability of the biliary tree, bile
  salt-independent bile flow and membrane fluidity.

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Phenothiazines

• In humans causes lesions reminiscent of
  immune-mediated hypersensitivity reactions in a
  small proportion of patients
• In dogs and Rhesus monkeys a reduction in bile
  flow occurs after its acute intravenous
  administration
• In isolated perfused rat liver chlorpromazine
  rapidly inhibits bile flow in a dose-dependent
  manner.
• Chlorpromazine inhibits Na/K ATPase activity.
• Chlorpromazine inhibits efflux of bile acids and
  indocyanine green from hepatocytes

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Other Agents Causing Cholestasis in Animals

• Lithocholic acid - actioncan be reversed by
  cholic acid suggesting a competition for
  transport proteins
• Ouabain Blocks Na/K pump
• Phalloidin and Cytochalasin B. Both affect actin
  microfilaments - possibly disrupting the actin
  corset around the bile canaliculus
• Cyclosporin A Causes symptoms of jaundice with
  no changes in the liver. Probably affects bile
  acid metabolism

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Toxic damage to bile duct lining cells

• Time course after treatment with alpha
  naphthyl-isothiocyanate When this is administered
  to rats at 300 mg/kg there is the following time
  course of events-
• 4h Bile duct dilation, loss of microvilli, tight
  junctions open
• 6h Similar but more pronounced, dilation of er
  and nuclear membrane of BDLCs. Some portal
  oedema.
• 8h Damaged bile ducts, cells exfoliating into
  duct.
• 24h Almost total destruction of bile ducts.
  Focal necrosis in the parenchyma
• 48h on Regeneration leading to complete repair

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ANIT Toxicity
8h
6h
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Mechanisms and Implications

• ANIT treatment affects only the middle and large
  sized ducts. These are the ducts where
  secretin-regulated water and ion transport occur.
  the damage caused by ANIT and similar compounds
  is probably due to the concentration of a toxin
  rising as water leaves the bile
• Repair of bile duct damage is even more rapid
  than repair of damage to hepatocytes. This rapid
  repair occurs in spite of apparently total
  destruction.

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Chronic Damage to Bile Ducts

• This may be seen both as a result of chemicals
  which affect the bile duct lining cells and as a
  spontaneous lesion in aging rats, probably
  associated with a viral infection
• Chronic treatment with ANIT results in
• 1) At 3 days there is acute damage to bile ducts
• 2) By 10 days there is proliferation of bile
  ducts surrounded by highly vascularised loose
  connective tissue
• 3) By 21 days there is proliferation of bile
  ducts which are now surrounded by mature
  connective tissue
• The final appearance of the spontaneous lesion is
  the same as the induced one

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Bile Duct Proliferation

• Two types may be present
• 1) Small fingers of cells push out into the
  surrounding parenchyma. In some cases the cells
  appear to be arranged in duct-like structures, in
  other cases not. This was associated with
  carcinogenesis but is more probably explained as
  repair by re-differentiation. The proliferating
  cells are termed oval cells
• 2) As previously described, portal tracts
  enlarge and are filled with large numbers of
  ducts which do not invade the parenchyma. There
  is no association between this and cancer

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Cholangiocarcinoma

• Liver tumours may have morphologies similar to
  hepatocytes or ducts, the latter are termed
  cholangiomas is benign, cholangiocarcinoma if
  malignant
• In some cases (eg coumarin) cholangioma is
  associated with bile duct proliferation, in
  others (eg DAB - dimethyl aminoazobenzene) with
  hepatocellular damage
• Both hepatocellular carcinomas and
  cholangiocarcinomas may be observed in a single
  study with agents such as DAB. It is not clear
  whether this means that oval cells are being
  targetted or that there is metaplasia of
  hepatocytes