MECHANI SMS OF DI SEASE

Vol ume 339 Number 17

1217

Review Article

Mechanisms of Disease

F

RANKLI N

H. E

PSTEI N

, M. D. ,

Editor

M

OLECULAR

P

ATHOGENESIS
OF

C

HOLESTASIS

M

ICHAEL

T

RAUNER

, M.D., P

ETER

J. M

EIER

, M.D.,

AND

J

AMES

L. B

OYER

, M.D.

From the Division of Gastroenterology and Hepatology, Department of
Medicine, Karl Franzens University, Graz, Austria (M.T.); the Division of
Clinical Pharmacology and Toxicology, Department of Medicine, Univer-
sity Hospital, Zurich, Switzerland (P.J.M.); and the Department of Medi-
cine and Liver Center, Yale University School of Medicine, New Haven,
Conn. (J.L.B.). Address reprint requests to Dr. Boyer at the Yale University
School of Medicine, P.O. Box 208109, New Haven, CT 06520-8019.
1998, Massachusetts Medical Society.

HE formation of bile is a vital function, and
its impairment by drugs or infectious, auto-
immune, metabolic, or genetic disorders re-
sults in the syndrome commonly known as cholesta-
sis.

1

The secretion of bile normally depends on the
function of a number of membrane transport sys-
tems in hepatocytes and bile-duct epithelial cells
(cholangiocytes) and on the structural and function-
al integrity of the bile-secretory apparatus. This re-
view summarizes the molecular defects in hepatocel-
lular membrane transporters that are associated with
various forms of cholestatic liver disease in humans.

MOLECULAR MECHANISMS OF BILE
FORMATION

Bile formation is an osmotic secretory process that
is driven by the active concentration of bile salts and
other biliary constituents in the bile canaliculi.

2

The
transport of solutes from the blood to the bile is
driven by transport systems in the plasma membrane
of the basolateral (sinusoidal) and apical (canalicu-
lar) surfaces of hepatocytes. The transport systems
most relevant to bile formation in the human liver
are illustrated in Figure 1 and listed with their cor-
responding functions in Table 1.
The basolateral plasma membrane contains the
Na

+

/K

+

ATPase that maintains the physiologic ex-
tracellular and intracellular ion gradients (more so-
dium outside the cell than inside; more potassium
inside than outside). In addition, Na

+

/K

+

ATPase,
together with a potassium channel, helps to generate
T

a transmembrane electrical potential of approximate-
ly 35 mV. These chemical and electrical potentials
are used for the maintenance of intracellular ion and
pH homeostasis. They provide the driving forces for
proton extrusion by a mechanism of sodiumhydro-
gen exchange and for bicarbonate entry by a mech-
anism of sodiumbicarbonate symport, as well as for
the electrogenic sodium-dependent uptake of conju-
gated bile salts (or bile acids).

4

Bile salts are the most
abundant solutes in bile. Their transport from plas-
ma into hepatocytes is predominantly mediated by
the sodiumtaurocholate cotransporter (NTCP).

5

In
contrast to conjugated bile salts, the unconjugated
bile salt cholate, the organic anion sulfobromophthal-
ein, and numerous other lipophilic albumin-bound
compounds are transported from plasma into hepa-
tocytes by sodium-independent transport systems,
including the organic-aniontransporting polypep-
tide

6

(OATP) (Fig. 1 and Table 1).
Under physiologic conditions, active transport of
solutes across the canalicular membrane of hepato-
cytes represents the rate-limiting step in bile forma-
tion. This unidirectional concentrative step is driven
by an array of ATP-dependent export pumps that
belong to the ATP-binding cassette family of mem-
brane transporters (Fig. 1 and Table 1). The first of
these canalicular transporters to be localized and
characterized was the multidrug-resistance-1 P-gly-
coprotein (MDR1), which mediates the canalicular
excretion of bulky lipophilic cations (e.g., anticancer
drugs, calcium-channel blockers, cyclosporine A, and
various other drugs).

7,8

However, its physiologic role
in overall bile formation remains unclear, because its
level of expression in liver is relatively low and its lev-
el of endogenous substrate is not known. In con-
trast, a clear and liver-specific function in bile forma-
tion could be assigned to the multidrug-resistance-3
P-glycoprotein (MDR3; multidrug-resistance-2 P-gly-
coprotein in rodent livers) (Fig. 1 and Table 1). As
demonstrated in mice in which the gene for multi-
drug-resistance-2 P-glycoprotein has been deleted
and in transfected yeast, this P-glycoprotein is a phos-
pholipid transporter that translocates phosphatidyl-
choline from the inner to the outer leaflet of the
canalicular membrane, where it can be selectively ex-
tracted by intracanalicular bile salts and secreted into
bile as vesicles and mixed micelles.

9

Another important hepatobiliary export pump is
the canalicular multispecific organic-anion transport-
er, which is a canalicular isoform of the multidrug-
resistanceassociated protein (MRP2).

10,11

It medi-
ates ATP-dependent canalicular excretion of a wide
range of amphipathic anionic substrates, including leu-
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1218

October 22, 1998

The New Engl and Jour nal of Medi ci ne

Figure 1.

Transport Polarity of Normal Hepatocytes and Bile-Duct Epithelial Cells (Cholangiocytes).
There are two sinusoidal systems for bile-salt uptake in hepatocytes (upper panel, left-hand side) a sodiumtaurocholate cotrans-
porter (NTCP) and a sodium-independent organic-anion transporter (OATP). Sodium-dependent uptake of bile salts through the
NTCP is driven by an inwardly directed sodium gradient generated by Na

+

/K

+

ATPase and the membrane potential generated in
part by a potassium channel. In addition, the basolateral membrane contains a sodiumhydrogen exchanger and a sodiumbicar-
bonate symporter (upper panel, right-hand side). The canalicular membrane contains several ATP-dependent export pumps: the
multidrug-resistance-1 P-glycoprotein (MDR1), the phospholipid transporter multidrug-resistance-3 P-glycoprotein (MDR3), the can-
alicular multispecific-organic-anion transporter (MRP2 or cMOAT), and the canalicular bile-saltexport pump (BSEP or SPGP). In
addition, the canalicular membrane contains several ATP-independent transport systems, including a chloride channel (distinct
from the cystic fibrosis transmembrane regulator protein), a chloridebicarbonate anion exchanger isoform 2 (AE2) for secretion
of bicarbonate, and a glutathione (GSH) transporter.
Cholangiocytes (lower panel) contain a chloride channel that corresponds to the cystic fibrosis transmembrane regulator (CFTR)
and a chloridebicarbonate anion exchanger isoform 2 (AE2) for secretion of bicarbonate. Furthermore, cholangiocytes have ab-
sorptive functions for a variety of bile solutes, including bile salts, amino acids, and glucose. PL denotes phospholipid, OA

organic
anion, BS

bile salt, OC

+

organic cation, and A

anion (possibly GSH).
Na
+
/K
+
ATPase
K
+
Na
+
BS

K
+
channel
OC
+
PL
OA

BS

Hepatocyte
CI

H
+
AE2
GSH
transporter
GSH
Na
+
H
+
exchanger
Na
+
HCO
3

symporter
HCO
3

CFTR Cl

K
+
Na
+
Na
+
CI

channel
CI

OA

, BS

BS

Glucose
Amino acid
Cholangiocyte
Na
+
CI

HCO
3

AE2
OATP
NTCP
MDR1
MDR3
MRP2
SPGP
HCO
3

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MECHANI SMS OF DI SEASE

Vol ume 339 Number 17

1219

kotriene C4, glutathione-S conjugates, glucuronides
(e.g., bilirubin diglucuronide and estradiol-17

b

-glu-
curonide), and sulfate conjugates, and is responsible
to a large extent for the generation of bile flow in-
dependent of bile salts within the bile canaliculi.

12

Finally, an ATP-dependent transport process is
also involved in the canalicular secretion of bile salts.
However, despite the quantitative importance of bil-
iary bile salts, the definite molecular identification of
the canalicular bile-salt transporter of the mammali-
an liver has lagged behind that of the other canalic-
ular transporters mentioned above. Although the
canalicular ecto-ATPase has been proposed as a pos-
sible candidate,

13

other investigators have provided
evidence that the canalicular bile-salt transporter may
also be a transport protein of the ATPase-binding
cassette type.

14

This assumption has recently been
confirmed by full-length cloning of a P-glycoprotein,
known as the sister of P-glycoprotein (spgp), from
rat livers and its functional expression in the oocytes
of

Xenopus laevis

and in Sf 9 insect cells demonstrat-
ing marked ATP-dependent bile-salt transport.

15

To-
gether with the expression of the sister of P-glyco-
protein in pig livers,

16

these findings strongly indicate
that this transporter represents the canalicular bile-
salt export pump of the mammalian liver.
Besides primary active-transport processes at the
plasma membranes of the canaliculi, the formation
and final composition of canalicular bile depend on
several other less well defined mechanisms, includ-

*These transporters are members of the ATP-binding cassette family.
These transporters have been defined on the basis of functional evidence; they have not yet been cloned from the human liver.
A number of transport systems have been functionally characterized at the luminal membrane of cholangiocytes for reclaiming substances from bile,
including glucose, amino acids, and bile salts.

3

T

ABLE

1.

N

OMENCLATURE

, L

OCATION

,

AND

F

UNCTION



OF

H

EPATOCYTE



AND

C

HOLANGIOCYTE

M

EMBRANE

T

RANSPORTERS


I

NVOLVED



IN

B

ILE

S

ECRETION

.

N

AME

A

BBREVIATION

L

OCATION

F

UNCTION

Hepatocyte

Ion transporters
Sodiumpotassium ATPase Na

+

/K

+

ATPase Basolateral (sinusoidal) membrane Maintains physiologic sodium and potassium gradi-
ents (extracellular Na

+

> intracellular Na

+

; intracel-
lular K

+

> extracellular K

+

)
Potassium channel K

+

channel Basolateral (sinusoidal) membrane Determines membrane potential
Sodiumproton exchanger isoform 1 NHE1 Basolateral (sinusoidal) membrane Acid extruder, housekeeping gene for intracellular pH
and cell volume
Sodiumbicarbonate symporter Na

+

HCO

3

symporter
Basolateral (sinusoidal) membrane Acid extruder, facilitates bicarbonate entry into hepa-
tocytes and bile by way of the canalicular chloride
bicarbonate anion exchanger isoform 2
Chloridebicarbonate anion exchanger
isoform 2
AE2 Canalicular membrane Acid loader excretes bicarbonate into bile and stim-
ulates bile flow independent of bile salts
Chloride channel C1

channel Canalicular membrane Facilitates chloride entry into bile
Organic-solute transporters
Sodiumtaurocholate cotransporter NTCP Basolateral membrane Primary carrier for conjugated bile-salt uptake from
portal blood
Organic-aniontransporting polypep-
tide
OATP Basolateral membrane Multispecific carriers for sodium-independent uptake
of bile salts, organic anions, and other amphipathic
organic solutes from portal blood
Multidrug-resistance-1 P-glycoprotein* MDR1 Canalicular membrane ATP-dependent excretion of various organic cations,
xenobiotics, and cytotoxins into bile
Multidrug-resistance-3 P-glycoprotein
(phospholipid transporter)*
MDR3 Canalicular membrane ATP-dependent translocation of phosphatidylcholine
from inner to outer leaflet of membrane bilayer
Multidrug-resistanceassociated protein
(canalicular multispecific organic-
anion transporter)*
MRP2
(cMOAT)
Canalicular membrane Mediates ATP-dependent multispecific organic-anion
transport (e.g., bilirubin diglucuronide) into bile;
contributes to bile-saltindependent bile flow
Canalicular bile-saltexport pump*
(sister of P-glycoprotein)
BSEP
(SPGP)
Canalicular membrane ATP-dependent bile-salt transport into bile; stimulates
bile flow dependent on bile salts; spgp of rat liver
mediates ATP-dependent bile-salt transport, indi-
cating that it represents the canalicular bile-salt
transporter of the mammalian liver
Glutathione transporter GSH transporter Canalicular membrane Glutathione transport into bile; stimulates bile flow
independent of bile salts

Cholangiocyte

Ion transporters
Cystic fibrosis transmembrane regulator CFTR Apical (luminal) membrane Chloride channel; facilitates chloride entry into bile
Chloridebicarbonate anion exchanger
isoform 2
AE2 Apical (luminal) membrane Facilitates bicarbonate secretion into bile and contrib-
utes to bile flow independent of bile salts
Organic-solute transporters
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1220

October 22, 1998

The New Engl and Jour nal of Medi ci ne

ing canalicular exocytosis of transcytotic and sub-
canalicular vesicles,

2

the activities of numerous nu-
cleotidases and peptidases,

17

periodic contractions of
the bile canaliculi,

18

and various activities of electro-
lyte transporters and ion channels of hepatocytes or
bile-ductule epithelial cells.

3,19

The last-named are
closely associated with the biliary excretion of chlo-
ride and bicarbonate and include the chloride
bicarbonate anion exchanger isoform 2 in both hep-
atocytes and bile-duct epithelial cells

20

and the cystic
fibrosis transmembrane regulator (CFTR), a chlo-
ride channel on the luminal membrane of bile-duct
epithelial cells (Fig. 1 and Table 1).

21

Regulated ex-
pression of these and other bile-duct transporters
and channels contributes substantially to the daily
output of bile, and their functional deficiency might
be an important cause of cholestatic liver disease.

EXPERIMENTAL MODELS AND THEIR
CLINICAL CORRELATES

Several animal models of intrahepatic and obstruc-
tive cholestasis simulate human cholestatic diseases.
These disorders include sepsis-induced cholestasis (in
endotoxin-treated rats), oral-contraceptiveinduced
cholestasis and cholestasis of pregnancy (in ethinyl
estradioltreated rats), and extrahepatic biliary ob-
struction induced by ligation of the common bile
duct. The cholestatic effects of endotoxin and endo-
toxin-induced cytokines not only have a role in the
pathogenesis of sepsis-induced cholestasis, but also
may explain defects in hepatobiliary excretory func-
tion during total parenteral nutrition and in alcohol-
ic and viral hepatitis.

22

Many drugs (e.g., cyclospor-
ine A and chlorpromazine) also cause intrahepatic
cholestasis at the level of the bile canaliculus in both
humans and animals.

1


Despite their different causes, each of these dis-
eases results in marked functional impairment of
hepatocellular uptake and canalicular excretion of
bile salts and various other organic anions.

23-27

Cho-
lestasis results from impaired transport of these
compounds into bile and the loss of osmotic driving
forces for bile secretion.

MOLECULAR MECHANISMS
OF CHOLESTASIS

Hepatocellular Transporters

Decreased or even absent expression of specific
hepatocellular transport proteins has been found in
several clinical forms (Table 2) and experimental
models of cholestasis. These abnormalities explain
the impairment of transport functions, with a subse-
quent reduction in bile flow and the development of
cholestasis.
The discovery that familial disorders of cholestasis
may be related to mutations in genes controlling
hepatocellular transport systems known to be in-
volved in the formation of bile is rapidly bridging the
gap between basic science and clinical medicine. Pro-
gressive familial intrahepatic cholestasis is a severe
type of cholestatic liver disease that is inherited as an
autosomal recessive trait (Table 2).

39

The disease pre-
sents in infancy and results in progressive cholestasis
and liver failure. Three types of progressive familial

T

ABLE

2.

M

OLECULAR

C

HANGES



OF

H

EPATOCELLULAR

-T

RANSPORT

S

YSTEMS



IN

P

ATIENTS



WITH

C

HOLESTATIC

D

ISORDERS

.

D

ISEASE

M

OLECULAR

C

HANGE

C

OMMENTS

R

EFERENCES

Progressive familial intrahepatic
cholestasis
Type 1 Mutation in P-type ATPase Mapped to chromosome 18q2122; low se-
rum

g

-glutamyltransferase concentrations
Carlton et al.,

28

Bull et al.

29

Type 2 Absence of sister of P-glycoprotein Mutation of sister of P-glycoprotein gene
(chromosome 2q24); low serum

g

-gluta-
myltransferase concentrations
Strautnieks et al.

30

Type 3 Absence of multidrug-resistance-3
P-glycoprotein RNA and protein
Mutation of multidrug-resistance-3 P-glyco-
protein gene (chromosome 7q21); high se-
rum

g

-glutamyltransferase concentrations
Deleuze et al.,

31


de Vree et al.

32

Benign recurrent intrahepatic
cholestasis
Mutation in P-type ATPase Mapped to progressive familial intrahepatic
cholestasis 1 locus (18q2122)
Bull et al.,

29

Houwen et al.

33

Extrahepatic biliary atresia Decrease in sodiumtaurocholate
cotransporter RNA
Inverse correlation with serum bilirubin con-
centrations; increase after successful porto-
enterostomy
Shneider et al.

34

Primary sclerosing cholangitis Increase in organic-aniontrans-
porting polypeptide RNA
Kullak-Ublick et al.

35

Primary biliary cirrhosis Decrease in chloridebicarbonate
anion exchanger, isoform 2 RNA
and protein
Hepatocytes and cholangiocytes affected Prieto et al.,

36

Medina et al.

37

Biliary obstruction Increase in multidrug-resistance-1
and multidrug-resistance-3
P-glycoprotein RNA
Direct correlation with serum bilirubin con-
centrations
Nozawa et al.

38
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MECHANI SMS OF DI SEASE

Vol ume 339 Number 17

1221

intrahepatic cholestasis are just now being recog-
nized. Type 1, also known as Bylers disease and
described originally in an Amish family, is character-
ized by low serum

g

-glutamyltransferase concen-
trations, high serum bile-salt concentrations, normal
serum cholesterol concentrations, and low biliary
chenodeoxycholic bile-salt concentrations. This form
of progressive familial intrahepatic cholestasis has
been mapped by positional cloning to chromosome
18q2122.

28,40

Benign recurrent intrahepatic cholestasis, a recur-
rent cholestatic disorder in adults, has also been
mapped to the same region of chromosome 18q21
22,

33,40

leading to speculation that there may be a fa-
milial cholestasis gene that is responsible for both
disorders despite their different phenotypes and
prognosis. Indeed, recently a gene mutation in pa-
tients with progressive familial intrahepatic cholesta-
sis type 1 and benign recurrent intrahepatic chole-
stasis has been described.

29

This gene (called

FIC1

)
normally encodes a P-type ATPase that is expressed
predominantly in the small intestine as well as in the
liver and is likely to have an important role in the
enterohepatic circulation of bile acids. Its known
function is to transfer aminophospholipids from the
outer to the inner leaflet of the plasma-membrane
bilayer. Further studies of the functional role of this
P-type ATPase should contribute importantly to the
understanding of bile formation and cholestasis.
Patients with findings typical of progressive famil-
ial intrahepatic cholestasis type 1, but unrelated to
the original Byler family, have also been described in
isolated populations in the Middle East, Greenland,
and Sweden and are considered to have Bylers syn-
drome. Homozygosity mapping and linkage analysis
in a Middle Eastern family resulted in the identifica-
tion of a gene locus on chromosome 2q24.

30

This
disorder has been designated progressive intrahepat-
ic cholestasis type 2. The gene locus for the canalic-
ular bile-salt transporter has also been mapped to
the same region of chromosome 2,

30

and mutations
in this transporter gene may be responsible for
progressive familial intrahepatic cholestasis type 2
(Thompson RJ: personal communication) (Table 2).
In contrast to types 1 and 2, the third subtype,
progressive familial intrahepatic cholestasis type 3, is
characterized by high serum g-glutamyltransferase
concentrations, as well as by bile-duct proliferation
and inflammatory infiltrates in the portal areas. The
underlying defect is a mutation (a 7-bp deletion or
a point mutation) of the multidrug-resistance-3 gene
(Table 2), resulting in the complete absence of the
multidrug-resistance-3 P-glycoprotein in the liver of
these patients and a substantial decrease in biliary
phospholipid concentrations with normal canalicular
excretion of bile salts.
31,32
Phospholipids in bile nor-
mally protect bile-ductule epithelial cells from the
toxicity of bile salts by forming mixed micelles
9
; there-
fore, the marked decrease or absence of biliary phos-
pholipids may explain the presence of bile-duct inju-
ry in these patients.
Thus, progressive familial intrahepatic cholestasis
type 3 provides an important link between a hepato-
cellular (canalicular) transport defect and the devel-
opment of cholangiopathies. Many patients with neo-
natal cholestasis (e.g., extrahepatic biliary atresia and
bile-duct paucity syndromes) and adult cholangiopa-
thies and other cholestatic syndromes (e.g., primary
biliary cirrhosis, primary sclerosing cholangitis, and
vanishing-bile-duct disorders) now need to be reeval-
uated for possible defects in the multidrug-resist-
ance-3 gene and its product.
9
Patients with primary
biliary cirrhosis have normal concentrations of mul-
tidrug-resistance-3 P-glycoprotein messenger RNA
(mRNA),
41
suggesting that decreased expression of
this gene is not involved in its pathogenesis.
Although no defects associated with the multi-
drug-resistance-1 gene have been identified so far,
mutations in this gene would be expected to have
an indirect role in certain types of drug-induced
cholestasis. As a consequence of such a hypothetical
defect, hepatocellular accumulation of drugs could
result in cholestasis, because substrates of multi-
drug-resistance-1 P-glycoprotein, such as cyclospor-
ine A, inhibit transport of ATP-dependent canalicu-
lar bile salts
42,43
and organic anions in rats.
42
The DubinJohnson syndrome is caused by a
point mutation of the gene for the canalicular multi-
specific-organic-anion transporter (also called multi-
drug-resistanceassociated protein), resulting in the
absence of this protein in the livers of affected pa-
tients.
44,45
Although patients with the DubinJohnson
syndrome usually have hyperbilirubinemia rather than
cholestasis, this syndrome is yet another example of
the way in which a mutation of a hepatocellular-
transporter gene can impair biliary excretory func-
tion. This syndrome is characterized by abnormal
biliary excretion of endogenous conjugates (e.g., bil-
irubin diglucuronide and coproporphyrin I) and ex-
ogenous amphiphilic anionic conjugates (e.g., conju-
gates of sulfobromophthalein and of indocyanine
green and iopanoic acid, an oral cholecystographic
agent), which are normally excreted by the canalicu-
lar multispecific organic-anion transporter.
46,47
In addition to genetic defects of the hepatocellular
transport systems (see above), exposure to cholestatic
injury may also result in molecular changes in baso-
lateral and canalicular transport systems in patients
with acquired forms of cholestasis. The potential
mechanisms for these forms of cholestatic liver disease
include changes in the rate of gene transcription;
post-transcriptional changes in mRNA processing,
stability, and translational efficiency; impaired intra-
cellular sorting or targeting; impaired protein activa-
tion (e.g., by phosphorylation or dephosphorylation);
and increased protein degradation.
48
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1222 October 22, 1998
The New Engl and Jour nal of Medi ci ne
Molecular alterations of basolateral transport pro-
teins may contribute to the functional impairment of
bile formation by diminishing the hepatocellular up-
take of biliary constituents.
24-26
In addition, these al-
terations may serve to prevent further accumulation
of toxic biliary constituents (especially bile salts)
within hepatocytes. For example, concentrations of
basolateral sodiumtaurocholate cotransporter pol-
ypeptide mRNA are decreased in patients with ex-
trahepatic biliary atresia, and the concentrations
increase if biliary drainage is restored by a portoen-
terostomy (Kasai procedure).
34
The mRNA concen-
trations are inversely related to the serum total bili-
rubin concentrations,
34
suggesting that the retention
of biliary constituents may lead to down-regulation
of the sodiumtaurocholate cotransporter. The pro-
moter of the sodiumtaurocholate cotransporter
gene in rats contains sequences for several transcrip-
tional regulatory elements involved in cytokine sig-
naling, as well as for elements responsive to steroids
and bile salts, and bile salts can suppress the promot-
er activity of this gene in vitro.
49
Furthermore, the
administration of endotoxin in rats inhibits the ac-
tivity of critical transcription factors (e.g., hepato-
cyte nuclear factor 1) that normally regulate this pro-
moter, resulting in decreased gene transcription in
sepsis-induced cholestasis.
50
Gene expression of the organic-aniontransport-
ing polypeptide, in contrast, appears to be up-regu-
lated in humans with cholestatic liver disease (pri-
mary sclerosing cholangitis) in which concentrations
of organic-aniontransporting polypeptide mRNA
are increased and up-regulated when transfected
hepatocytes are exposed to bile salts in cells trans-
fected in vitro. This up-regulation might minimize
the accumulation of potentially toxic compounds by
transporting these substances out of the hepatocytes
into portal venous plasma.
35
Since the transport of biliary constituents across
the canalicular membrane is the rate-limiting step in
bile formation, the impairment of canalicular trans-
port systems should have a major role in the patho-
genesis of acquired forms of intrahepatic cholestasis,
similar to its role in hereditary defects (see above).
Decreased canalicular secretion of bile salts and a
broad range of anionic conjugates (e.g., bilirubin di-
glucuronide) is a fundamental pathophysiologic de-
fect in all forms of cholestasis.
23,25,27
Indeed, the mo-
lecular expression of the corresponding transport
systems, such as the bile-saltexport pump and the
canalicular multispecific organic-anion transporter, is
decreased in experimental models of cholestasis,
findings that may provide the molecular basis for im-
paired excretion of bile salts and the development of
jaundice in humans with cholestasis.
51,52
Expression of the chloridebicarbonate anion ex-
changer isoform 2 is reduced in the livers of patients
with primary biliary cirrhosis.
36,37
Since chloride
bicarbonate exchange activity contributes to the se-
cretion of both canalicular and ductular bile (Table
1), decreased hepatic expression of this transporter
could lead to impaired bile flow. Diminished expres-
sion of anion exchanger isoform 2 has also been re-
ported in the salivary glands of patients with primary
biliary cirrhosis and the sicca syndrome,
53
which may
indicate a more generalized epithelial failure of bicar-
bonate secretion. In contrast, concentrations of he-
patic MDR1 and MDR3 mRNA are increased in pa-
tients with obstructive cholestasis,
38
in whom such
increases are strongly correlated with increases in
serum bilirubin and alkaline phosphatase concen-
trations.
In summary, the altered expression of hepatocellu-
lar transport systems in human cholestatic liver disease
and experimental models of cholestasis
24-26,51,52,54-58
may provide a molecular correlate for the functional
changes that occur in cholestasis. Some of these
changes contribute to cholestasis, whereas others may
limit the accumulation of toxic biliary constituents in
hepatocytes.
Cholangiocyte Transporters
Knowledge about cholangiocyte transport func-
tion at the molecular level in cholestasis is more lim-
ited.
3
Mutations of the CFTR,
59
which is located on
the luminal membrane of cholangiocytes, but not
hepatocytes,
21
result in impairment of ductal secre-
tion of chloride and water. This defect is associated
with mucosal obstruction of the intrahepatic bile
ducts, leading to focal areas of biliary fibrosis and
cirrhosis in patients with cystic fibrosis.
60
OTHER STRUCTURAL AND FUNCTIONAL
DEFECTS: ALTERED CYTOSKELETON,
TIGHT JUNCTIONS, VESICULAR
TRANSPORT, AND SIGNAL
TRANSDUCTION
Most human and animal cholestatic liver disorders
are associated with profound changes in the cyto-
skeleton of the hepatocytes, including disruption of
microtubules, increases in intermediate filaments,
and accumulation of disorganized bundles of actin
microfilaments in the pericanalicular domain (Fig.
2).
18
These cytoskeletal changes result in the loss of
apical microvilli and diminished contractility of the
canalicular membrane and may also contribute to
the leakiness of tight junctions between cells.
61
Some forms of severe familial cholestasis, resulting
in cirrhosis in children in Canada (North American
Indian childhood cirrhosis), are associated with
large increases in pericanalicular microfilaments rem-
iniscent of but not identical to toxicity from phal-
loidin.
62
Cholestasis induced experimentally in rats is also
associated with the disruption of the structural and
functional integrity of the hepatocellular tight junc-
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MECHANI SMS OF DI SEASE
Vol ume 339 Number 17 1223
tions (Fig. 2).
61,63
This abnormality results in in-
creases in paracellular permeability, regurgitation of
biliary constituents into plasma, and reduction of
the osmotic gradients in the bile canaliculi that nor-
mally constitute the driving force for bile secretion.
The hepatic localization and expression of the tight-
junction proteins that normally form the junctional
barrier, such as zonula occludens 1 and occludin, are
altered in rats by ligation of the common bile duct
and treatment with ethinyl estradiol.
64-66
Aggregates
of zonula occludens 1 along the borders of the
canaliculi become distorted and accumulate in the
cytoplasm, and occludin no longer localizes with
zonula occludens 1, resulting in leaky junctions.
66
Targeting of membrane components, transcytosis,
and canalicular exocytosis of vesicles are also disrupt-
ed during cholestasis, resulting in the retention of
apical (canalicular) transporters on the basolateral
surface of hepatocytes and a delay in vesicle trans-
port to the bile canaliculi.
67-69
Accumulation of ves-
icles within the pericanalicular region of hepatocytes
is a characteristic morphologic finding in many
forms of cholestatic liver injury (Fig. 2).
70,71
High
concentrations of bile salts, such as chenodeoxychol-
ic acid, inhibit the function of molecular motors,
such as kinesin and dynein, that move vesicles along
microtubules.
72
Impairment of the movement of ves-
icles during cholestasis results in a decreased number
of functional transporters in the canalicular mem-
brane and thus contributes to cholestasis.
73
Calcium signaling within and between hepato-
cytes is impaired in cholestasis.
74,75
Gap-junction
proteins (connexins 32 and 26) disappear within 24
hours after ligation of the common bile duct in rats,
which results in a decline in the migration of calcium
waves between individual hepatocytes (Fig. 2).
75
This decline may diminish orderly contractions of
the bile canaliculi and impair the process of micro-
peristalsis that normally facilitates the movement of
bile from the terminal canaliculi toward the bile
ductules in the portal tracts counter to the direction
of blood flow.
76
Cyclic-AMPmediated intracellular signaling in
hepatocytes is also impaired after ligation of the com-
mon bile duct, as a consequence of altered expres-
sion and subcellular localization of heterotrimeric
G proteins. These changes, together with changes in
the composition of liver-membrane lipids and the
detergent effect of bile salts, may contribute to the
impairment of adenylate cyclase activity
77,78
and
decrease the stimulatory effects of glucagon and vas-
oactive intestinal peptide on bile secretion.
77,78
In
contrast, in cholangiocytes, secretin-receptor gene
expression is up-regulated after ligation of the com-
mon bile duct
79
and may contribute to the increased
choleretic effect of secretin that follows the prolifer-
ation of bile ductules in rats. Up-regulation of bicar-
bonate secretion by cholangiocytes, together with
down-regulation of the canalicular multispecific or-
ganic-anion transporter that excretes bilirubin, may
account for the well-known clinical complication of
white bile that occurs during prolonged obstruc-
tion of the bile ducts.
THERAPEUTIC IMPLICATIONS AND
FUTURE PERSPECTIVES
Pharmacologic Therapy
Ursodiol (ursodeoxycholic acid) is currently the
accepted therapy for patients with primary biliary
cirrhosis, and it may extend life by slowing the pro-
gression of disease, according to the results of three
trials.
80
It may also have beneficial effects in certain
other cholestatic liver disorders, including primary
sclerosing cholangitis, intrahepatic cholestasis of preg-
nancy, and cystic fibrosis.
81
However, large trials have
not been conducted among patients with these dis-
eases, and a recent small, randomized study of pri-
mary sclerosing cholangitis showed no benefit of ur-
sodiol with regard to survival.
82
Several potential molecular mechanisms may ac-
count for the beneficial actions of ursodiol. It replac-
es toxic hydrophobic bile salts in serum, liver, and
bile.
81,83
Conjugated ursodiol is adsorbed to the in-
terface of the plasma membrane at the extracellular
space, where it may prevent the extraction of mem-
brane lipids by more hydrophobic bile salts.
84
In pa-
tients with defective biliary phospholipid secretion
(progressive familial intrahepatic cholestasis type 3),
the beneficial effect of ursodiol may be related to its
enrichment in bile and the modulation of biliary
bile-salt composition in favor of hydrophilic bile
salts, which are not toxic to the biliary epithelium,
despite the absence of phospholipids in bile.
81,85
Ur-
sodiol also down-regulates expression of abnormal
major histocompatibility complex (MHC) class I
molecules in periportal hepatocytes of patients with
primary biliary cirrhosis, whereas expression of ab-
normal MHC class II molecules on bile-duct epithe-
lial cells does not change.
86
However, it is unclear
whether these effects represent specific immuno-
modulatory properties of ursodiol
87
or are due to
improvement of the cholestatic liver injury, which
also increases expression of MHC class I molecules
on hepatocytes.
88
Experiments in rats indicate that the taurine con-
jugate of ursodeoxycholic acid, the principal form of
ursodeoxycholic acid in the body, may increase the
excretory capacity of cholestatic liver cells by stimu-
lating apical exocytosis.
74,89,90
This increase, in turn,
should induce targeting and insertion of transport
proteins into the canalicular membrane and increase
the capacity to excrete more hydrophobic bile salts
into bile, thereby reducing liver-cell injury.
90
The re-
sults of kinetic analysis of biliary excretion of syn-
thetic derivatives of bile salts in patients with primary
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1224 October 22, 1998
The New Engl and Jour nal of Medi ci ne
B
Normal Hepatocyte
1. Loss of gap junctions
2. Leaky junctions
3. Disorganized actinmyosin
bundles
4. Disruption of transcytotic
vesicular pathway
Cholestatic Hepatocyte
A
4
3
1
2
4
Water
3
1
2
1. Gap junction
2. Tight junction
3. Pericanalicular actinmyosin
network
4. Transcytotic vesicular pathway
(microtubule-dependent)
Water
Downloaded from www.nejm.org on October 12, 2008 . Copyright 1998 Massachusetts Medical Society. All rights reserved.
MECHANI SMS OF DI SEASE
Vol ume 339 Number 17 1225
biliary cirrhosis and primary sclerosing cholangitis
support the view that therapy with ursodiol increases
the capacity of the liver to excrete bile salts.
91
Up-
regulation of the expression of the chloridebicar-
bonate anion exchanger isoform 2 in patients with
primary biliary cirrhosis who were treated with urso-
diol has also been reported.
36,37
It also directly stimu-
lates chloride secretion in human gallbladder cells by
activating a calcium-sensitive chloride channel, an ef-
fect that might be of benefit in patients with cystic
fibrosis.
92
Gene Therapy
The retrograde infusion of an adenovirus encod-
ing the human CFTR into the common bile duct of
rats resulted in the temporary expression of CFTR
protein.
93
In addition, this defect has been tempo-
rarily corrected in vitro in isolated bile-duct cells
from two patients with cystic fibrosis, again using an
adenovirus-based vector.
94
These approaches open
new horizons in the treatment of patients with cyst-
ic fibrosis by delivering encoding vectors for the
CFTR through endoscopic retrograde cholangiog-
raphy.
95
CONCLUSIONS
The rapid advances in the understanding of the
cellular and molecular physiology of bile secretion
have led to a better grasp of the pathophysiology of
and structural cell damage caused by various hered-
itary and acquired cholestatic disorders. These ad-
vances should lead to the development of more ef-
fective preventive measures and new therapeutic
strategies for a whole variety of currently untreatable
cholestatic liver diseases.
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Figure 2. Cell Contacts, Cytoskeleton, and Vesicular Targeting
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