Diseases of the Gallbladder and Bile Ducts - part 9 potx

• Recognize the major hepatic transporters responsible for the sinusoidal uptake of bile salts or organic anions or the efflux of bile salts, organic anions, or lipids into bile • Underst

with PBC who are receiving corticosteroids [48] However,

in a randomized, double-blind, placebo-controlled study in

67 patients with PBC, etidronate did not cause any

improve-ment of bone mass density (BMD) A more recent study,

in-cluding 32 female patients with PBC, showed that both

etidronate and alendronate increase BMD but the positive

effect of alendronate was far superior [49] So, most likely

alendronate, rather than etidronate, should be

recommen-ded to patients with PBC with osteoporosis, but further

stud-ies are required to establish the role of this agent in the

treatment of osteoporosis in PBC It cannot be ignored that in

patients with advanced PBC (who are also more likely to have

osteoporosis) and who have esophageal varices,

biphospho-nates may potentially cause esophagitis and increase the risk

of variceal bleeding

Vitamin K plays a modulatory role on bone metabolism

Increased BMD and prevention of bone fractures were

ob-served in patients with osteoporosis who were treated with

vitamin K A randomized study in female patients with PBC

showed a signifi cant increase of BMD in subjects treated with

vitamin K [50] These results are promising but require

con-firmation in studies including larger cohorts of patients

After publication of the results of the HERS II trial (heart

and estrogen/progestin replacement study) HRT cannot be

recommended anymore for the treatment of osteoporosis as

it increases the risk of certain malignancies, hip fracture, and

thromboembolism and does not have any signifi cant

cardio-protective effect Also UDCA and calcitonin seem to be of no

use in the prevention or treatment of osteoporosis in patients

with PBC

A signifi cant proportion of patients with PBC is deficient of

fat soluble vitamins This refers mostly to patients with

ad-vanced disease, in particular in those who also have low

serum albumin and cholesterol levels and an elevated

biliru-bin When the Mayo risk score for PBC is higher or equal to 5,

patients may be found to be vitamin A deficient It has been

recommended that these patients should be screened for

fat-soluble vitamin deficiencies and adequately supplemented if

necessary

As with all patients who are found to have large

eso-phageal varices, prophylactic nonselective beta blockade is

recommended

Specific therapies for PBC

There is no medical therapy that cures PBC Ursodeoxycholic

acid (UDCA) remains the only US Food and Drug

Adminis-tration approved medication for PBC Although the

mecha-nisms involved in its hepatoprotective properties have been

extensively studied over last 20 years, the mechanisms of its

action are not yet fully explained UDCA and its conjugates

stimulate bile salt excretion and protect against

bile-salt-induced mitochondrial damage, oxidative stress, and

apop-tosis and also may have a membrane-stabilizing effect,

protecting against bile-salt-induced solubilization There is

no doubt that UDCA plays the role of a signaling molecule with precise regulatory properties in specific signaling pathways [51] UDCA causes signifi cant improvement in biochemical parameters of cholestasis, including bilirubin, alkaline phos-phatase, and GGT It also decreases serum cholesterol levels UDCA slows down the histological progression of the disease [52,53] It may also decrease pruritus in a proportion of pa-tients (but rarely it may increase pruritus) but has no signifi -cant effect on fatigue A beneficial effect on survival was observed when patients were treated for up to 4 years with the trial dosage of UDCA [54], although clearly some patients respond better than others It has been shown that those pa-tients with noncirrhotic PBC treated with UDCA appear to have a 10-year survival that is no different from an age- and gender-matched population [55] Overall the greatest effect

on short-term survival was seen in those patients with more severe disease, for whom UDCA treatment may delay the need for transplantation Treatment before transplant does not have a detrimental effect on the patients’ post-transplant outcome, despite the patients being older when they eventu-ally come to need a transplant At the recommended dose of

13 to 15 mg/kg per day UDCA is extremely well tolerated with only a minority of patients complaining of diarrhea – but lower doses, that is 10 mg/kg, appear to be less effective.The beneficial effect of UDCA has been questioned [56] and no signifi cant impact of this drug on survival or time to liver transplantation was found, although a signifi cant re-duction in jaundice and ascites was noted This report has been criticized as early studies, where patients were treated for short periods of time and with subtherapeutic doses of UDCA, were included in the meta-analysis

Although the trigger for the development of PBC remains

to be elucidated, autoimmune phenomena may play a role

in the hepatic damage Thus different immunosuppressive drugs have been investigated in PBC In a study published by Christensen et al, azathioprine was found to prolong survival

by approximately 20 months [57] A positive effect on vival was also observed by another European group, in a pla-cebo controlled trial with cyclosporine A [58] Unfortunately, this was associated with signifi cant side-effects of this calci-neurin inhibitor, including renal impairment and hyperten-sion Anecdotal reports suggest that combined therapy of UDCA and mycophenolate mofetil (MMF) may be of partic-ular use in patients with a signifi cant infl ammatory compo-nent on their biopsies The potential application of MMF in PBC is now the subject of an ongoing randomized study With regard to other treatments, no convincing effect was observed with colchicine and methotrexate Budesonide was found to increase the risk of portal vein thrombosis in cirrhotic pa-tients with PBC [59] and therefore its use in this subgroup of PBC patients should be discouraged Drugs more recently in-vestigated in PBC include bezafibrate, pranlukast and sulin-dac [60] Their role in the management of PBC remains to be established

sur-As PBC progresses very slowly, it is extremely difficult to

prove effi cacy of any therapeutic agent It has been suggested

that in order to demonstrate a clear effect of any medication

on death rate in patients without liver transplantation, over

200 patients have to be treated for a period of 5 years [58] For

those who reach end-stage liver disease, liver

transplanta-tion is the only optransplanta-tion but, certainly, liver transplantatransplanta-tion

can not be reliably used as a primary end-point

The proportion of patients with PBC who require liver

transplantation, either for end-stage liver failure or for poor

quality of life, is diminishing [61] PBC used to be the most

common indication for liver transplantation in patients with

end-stage liver disease, comprising up to 55% of all

trans-planted, cirrhotic patients in some centers [45] This

propor-tion has now decreased to 10%; this is in part due to both

increasing number of patients being transplanted for viral-

and alcohol-related cirrhosis and possibly a protective effect

of UDCA [45] Indications for liver transplantation in PBC

are summarized in Table 21.4 The Mayo Clinic model and

the more recently introduced MELD score are useful in

pre-dicting survival in patients with end-stage liver disease It is

recommended that in patients with PBC, once their bilirubin

level has reached 100 µmol/L (5.9 mg/100 mL), they should

be referred for liver transplant assessment [45] Current

5-year survival after liver transplantation for PBC varies

between 83 and 86%, making this disease an excellent

indi-cation for grafting Early mortality after surgery is caused

mostly by multiorgan failure and sepsis [61] Chronic

rejec-tion, which is the most common indication for regrafting

within 1 year of surgery, occurs signifi cantly less commonly

in patients with PBC than in patients transplanted for immune hepatitis and more commonly than in those trans-planted for alcohol-related liver disease [62] PBC does recur after transplantation and the diagnosis of the recurrence can only be reliably established by histological examination [45] This phenomenon is rarely of clinical signifi cance and may potentially be associated with tacrolimus rather than cyclosporine-based immunosupression [63]

auto-A vital issue, which has to be addressed in the near future,

is the identifi cation of patients who are more likely to ress into end-stage liver disease Clinical practice clearly shows that some subjects develop cirrhosis despite being treated with UDCA whereas others show no clinical or histo-logical progression over many years, sometimes even with no treatment The role of newly identified serum markers of dis-ease progression has to be validated Certainly, genetics may play a crucial role in the natural history of this disease, affect-ing the rate of progression and response to treatment This di-lemma has prompted several leading groups from all over the world to establish a collaborative project which will allow complex genetic analyses in a large, shared pool of samples Undoubtedly, this effort will enable an unprecedented accel-eration in our knowledge on the genetic background of PBC and may have a signifi cant impact on combating this chronic and potentially lethal disease

prog-Questions

1 Which sentence is true about the epidemiology of PBC?

a recent studies have shown that the prevalence of PBC is decreasing

b smoking does not increase the risk for developing PBC

c the rate of AMA seropositivity in the general population is significantly higher than the prevalence of PBC arising from epidemiological studies

d all these statements are false

e statements (b) and (c) are true

2 In the pathogenesis of PBC

a there is a clear relationship to herpes zoster infection

b apoptosis may play a role

c N aromaticivorum may play a role in triggering the disease

d there is a very weak association with possible genetic factors

e statements (b) and (c) are true

3 With regard to the natural history of PBC which of the following

statements is true?

a the majority of patients diagnosed today are jaundiced

b pruritus never precedes the onset of jaundice

c the asymptomatic phase usually lasts a couple of weeks

d AMA-positive patients who have normal biochemistry may have features of PBC on their biopsies

e the onset of the disease is usually acute

Table 21.4 Indications for transplantation in patients with primary

Increasing muscle wasting

Increasing symptomatic osteopenia

Encephalopathy

Intractable ascites

Recurrent, intractable variceal hemorrhage

Spontaneous bacterial peritonitis

Moderate hepatopulmonary syndrome

Early hepatocellular carcinoma

Biochemical

Serum bilirubin > 170 µmol/L > 6 months

Serum albumin < 25 g/L

Patients should be referred to a liver transplant center when their

bilirubin reaches 100 µmol/L (Reproduced from MacQuillan GC and

Neuberger J Clin Liver Dis 2003;7:941–56, with permission from

Elsevier.)

4 In the natural history of PBC

a factors predisposing to progression from asymptomatic to

symptomatic disease include bilirubin and alkaline

phosphatase

b as many as 90% of initially asymptomatic patients may develop

symptoms of liver disease within 4 years of diagnosis

c PBC in children usually has a very aggressive course

d the diagnosis of PBC is most commonly established by the age

of 20

e all statements are false

5 Fatigue in PBC – which statement is true?

a the origin of fatigue in PBC is most likely central

b fatigue is a rare symptom in PBC

c there is a strong correlation between age and degree of

fatigue

d there is a strong correlation between fatigue and hepatic

histology

e fatigue in PBC is usually relieved by rest

6 Pruritus in PBC – which statement is true?

a pruritus occurs in about 30% of patients

b pruritus is usually worst in the morning

c the palms or soles are never affected

d the classical “butterfly” area delineates the area of most intense

scratching

e scratching usually does not relieve this symptom

7 Diseases associated with PBC – which statement is true?

a sicca syndrome occurs in approximately 10% of patients

b a history of hypothyroidism is present in 90% of patients

c psoriasis is the commonest associated disease

d majority of patients are found to have antiendomysial

antibodies

e all statements are false

8 Diagnosis of PBC – which statement is true?

a diagnosis is established on the presence of positive

ANA

b AMA may be positive in about 40 to 50% of patients

c liver biopsy is essential for establishing the diagnosis

d immunochemistry for AMA may be falsely positive in patients

with type 2 autoimmune hepatitis

e the presence of AMA confirms only advanced PBC

9 Treatment of PBC – which statement is true?

a the curative rate of ursodeoxycholic acid is approximately

60%

b questran should be avoided in the treatment of pruritus

c rifampicin is a first-line treatment of pruritus

d HRT is highly recommended to prevent osteoporosis only in

postmenopausal women with PBC

e budesonide must not be used in cirrhotic patients with

PBC

10 Liver transplantation in PBC – which statement is true?

a amongst patients with cirrhosis, PBC is currently the most common indication for liver transplantation

b the 5-year survival after liver transplantation for PBC is approximately 85%

c treatment with ursodeoxycholic acid before transplant significantly reduces survival following liver transplantation

d calcineurin inhibitors (tacrolimus and cyclosporin A) have to be avoided after liver transplantation as they increase the risk of acute rejection

e recurrence of the disease is the most challenging issue after liver transplantation

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15 Mato TK, Davis PA, Odin JA, et al Sidechain biology and the

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16 Mendel-Hartvig I, Nelson BD, Loof L, Totterman TH Primary

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17 Selmi C, Balkwill DL, Invernizzi P, et al Patients with primary

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18 Vergani D, Bogdanos DP, Baum H Unusual suspects in primary

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19 Abdulkarim AS, Petrovic LM, Kim WR, et al Primary biliary

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20 Xu L, Sakalian M, Shen Z, et al Cloning the human

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21 Xu L, Shen Z, Guo L, et al Does a betaretrovirus infection trigger

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22 Mason AL, Farr GH, Xu L, et al Pilot studies of single and

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23 Leung PS, Quan C, Park O, et al Immunization with a

xeno-biotic 6-bromohexanoate bovine serum albumin conjugate

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24 Sasaki M, Ansari A, Pumford N, et al Comparative

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25 Kimura Y, Selmi C, Leung PS, et al Genetic polymorphisms

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26 Brind AM, Bray GP, Portmann BC, Williams R Prevalence and

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27 Jones DE, Watt FE, Metcalf JV, et al Familial primary biliary

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28 Selmi C, Mayo MJ, Bach N, et al Primary biliary cirrhosis in

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29 Prince M, Chetwynd A, Newman W, et al Survival and

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33 Vleggaar FP, van Buuren HR, Zondervan PE, et al Jaundice in non-cirrhotic primary biliary cirrhosis: the premature ducto- penic variant Gut 2001;49:276–81.

34 Dahlan Y, Smith L, Simmonds D, et al Pediatric-onset primary biliary cirrhosis Gastroenterology 2003;125:1476–9.

35 Dickson ER, Grambsch PM, Fleming TR, et al Prognosis in mary biliary cirrhosis: model for decision making Hepatology 1989;10:1–7.

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38 Rudi J, Schonig T, Stremmel W Therapy with ursodeoxycholic acid in primary biliary cirrhosis in pregnancy Z Gastroenterol 1996;34:188–91.

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45 MacQuillan GC, Neuberger J Liver transplantation for primary biliary cirrhosis Clin Liver Dis 2003;7:941–56.

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Immuno-S E C T ION 3.3

Intrahepatic cholestasis

Copyright © 2006 by Blackwell Publishing Ltd

• Recognize the major hepatic transporters responsible for the sinusoidal uptake of bile salts or organic anions or the efflux

of bile salts, organic anions, or lipids into bile

• Understand how transcription factors co-ordinate the response of hepatocyte transporters and detoxification enzymes to cholestatic liver injury

• Learn about the major genetic causes of cholestatic diseases in children and adults

• Identify the clinical features and molecular mechanisms for drug-induced cholestatic liver injury

Multiple etiologies are responsible for cholestatic liver injury,

which require vastly different diagnostic and therapeutic

ap-proaches In this chapter, we will review a diverse group of

nonsurgical diseases that can mimic the presentation of other

causes of cholestatic liver disease discussed elsewhere in this

book In order to understand how these diverse disorders can

cause cholestatic liver injury, we will concisely review the

current understanding of the key proteins that constitute the

normal hepatic uptake and biliary excretory pathways In

human disease and animal models, the molecular regulation

of these key transport proteins in response to cholestatic liver

injury has provided detailed insight into the

pathophysiolog-ical mechanisms in different cholestatic conditions Clinpathophysiolog-ical

presentation, evaluation, treatment, and medical conditions

associated with cholestasis will follow a review of the normal

biliary physiology A review of drug-induced cholestasis will

also be provided, as inability to recognize this important

eti-ology can lead to persistent exposure to the offending agent

and unnecessary diagnostic or therapeutic interventions We

anticipate that enhanced understanding of molecular

mech-anisms of cholestatic liver disease should lead to new

thera-peutic strategies for these widely different diseases in the

future

Mechanisms for hepatic bile formation

The cellular organization of the liver combined with the

unique endothelial structure of the sinusoids are ideally

suit-ed for the efficient sinusoidal uptake and effl ux of molecules

by the hepatocytes as well as their biliary excretion into the

canaliculus, the most proximal portion of the biliary system

355

[1–4] The hepatocytes, like other epithelial cells such as the enterocyte, are polarized cells with distinct membrane do-mains exposed to either the sinusoidal surface, referred to as the basal or sinusoidal domain, or to the canaliculus, referred

to as the apical or canalicular domain The membrane tween these regions forms the lateral domain Cords of hepa-tocytes two cell thick are attached at their sinusoidal and apical domain by gap junctions, generating a physical barrier between the vascular sinusoidal space and the biliary excre-tory pathway Ions and water can travel between these gaps and disruption of these anchors between hepatocytes can lead to regurgitation of biliary components into the vascular space during cholestatic conditions Bile formation requires both the maintenance of these cell-to-cell contacts as well as vectorial uptake at the sinusoidal membrane, followed by se-cretion of biliary components at the canalicular membrane.Based on animal studies, the origins of bile flow are divid-

be-ed into bile acid dependent and independent bile flow Bile salts are the predominate solute in bile which, along with sodium ion, are responsible for water movement into the canalicular space, predominately by paracellular pathways between adjacent hepatocytes Bile salts are also essential for biliary excretion of phospholipids, which are extracted from the outer leaflet of the canalicular membrane forming mixed micelles in the presence of bile salts An absence of bile salts leads to signifi cant reduction in bile formation and phospho-lipid excretion On the other hand, bile salt excretion into the canalicular space in the absence of phospholipid excretion leads to extensive damage to cholangiocytes, due to potent, detergent and cytotoxic effects of free bile salts A bile-salt-independent bile flow also exists in which effl ux of organic

Copyright © 2006 by Blackwell Publishing Ltd

anions and the important peptide antioxidant, glutathione

(GSH), coupled with its cleavage by γ glutamyl

transpepti-dase and dipeptitranspepti-dases into its component amino acids,

gener-ate an osmotic gradient promoting wgener-ater movement into the

canalicular space [5] Bicarbonate secretion is another

im-portant component whose contribution is highly species

de-pendent [6]

The bile canalicular space created by adjacent hepatocytes

eventually empty at the periphery of the lobule into ductules

lined by cholangiocytes which further modify ion and

bicar-bonate content in bile, which is regulated by secretin A

cho-lehepatic shunt between the hepatocytes and cholangiocytes

has been proposed to explain the bicarbonate-rich biliary

ex-cretion caused by infusion of unconjugated bile acids such as

ursodeoxycholate in animals The role this plays in normal

bile flow is controversial It is postulated that certain

uncon-jugated bile acids secreted by hepatocytes are reabsorbed by

cholangiocytes in protonated form (leaving bicarbonate

be-hind) and then returned to the hepatocytes via the ductular capillaries, emptying into the sinusoids in a repetitive recy-cling, leading to enhanced bicarbonate secretion associated with a hypercholeresis [7] In addition to fl uid and electro-lyte movement, mechanical contraction of canalicular membrane by a microfilament network located at the apical domain of hepatocytes also promote bile flow by a “squeezing action” on the canalicular space

Within the last decade, identifi cation of cellular transport proteins mediating sinusoidal uptake and biliary secretion of the key solutes has greatly expanded our understanding of the cellular mechanism for bile formation and its dysregula-tion in cholestatic conditions We will review the key trans-port proteins listed in Table 22.1 and illustrated in Fig 22.1 at each domain and the nuclear receptors that regulate their ex-pression The altered regulation of these transporters in re-sponse to cholestatic stimuli are now known to play a key role

in the development and maintenance of cholestasis [3,4]

Table 22.1 Molecular and functional characteristics of human hepatocyte membrane transporters.

Organic anion transport peptide OATP1B1 SLCO1B1 Sinusoidal Bi Bile salts, HMG-Co reductase inhibitors,

Organic anion transport peptide OATP1B3 SLCO1B3 Basolateral Bi Bile salts, digoxin

(OATP-8)

Organic anion transport peptide OATP2B1 SLCO2B1 Basolateral Bi Estrone-3-sulfate,

Bile salt export pump BSEP ABCB11 Canaliculus Uni Taurocholate, bile acids

ABCG5/ABCG8 ABCG5/ABC G8 ABCG5/ Canaliculus Uni Cholesterol

ABCG8

Multidrug resistance 3 MDR3 ABCB4 Canaliculus Uni Phosphatidylcholine

Familial intrahepatic FIC1 ATP8B1 Canaliculus Uni Unknown

cholestasis 1

Multiresistance protein 2 (cMOAT) MRP2 ABCC2 Canaliculus Uni Leukotriene C4, GSH conjugates, conjugated

Multiresistance protein 3 MRP3 ABCC3 Basolateral Uni Bile acids, conjugated sex steroids

Multiresistance protein 4 MRP4 ABCC4 Basolateral Uni Nucleotide analogs, organic anions, bile

salts (with GSH) Multiresistance protein 5 MRP5 ABCC5 Basolateral Uni Nucleotide analogs, organic anions

Multiresistance protein 6 MRP6 ABCC6 Basolateral Uni Organic anions

Location (domain) of human hepatic transporters and some of their model substrates [3,21,28] Transport features refers to the ability of the

transporter to function as a unidirectional (Uni) or bidirectional (Bi) transporter.

Sinusoidal membrane

Uptake of bile acids by the sinusoidal membrane is a key step

in the enterohepatic circulation of bile salts in which

approx-imately 95% of the bile salt pool is efficiently recovered by the

intestine and resecreted by the liver [8] Initial

characteriza-tion of bile acid and bile salt uptake in isolated hepatocytes

and enriched sinusoidal plasma membrane vesicles

identi-fied both sodium dependent and independent transport

sys-tems for bile salts In addition, hydrophobic secondary bile

acids or unconjugated bile acids may also enter into the

hepa-tocytes by passive diffusion Using an expression cloning

strategy, a specific sodium-dependent bile salt transporter

has been identifi ed as well as sodium-independent organic

anion transporters, some of which can also mediate

sodium-independent bile acid uptake

Sodium-dependent bile acid transporter

The sodium-dependent taurocholate carrier protein, Ntcp, is

a 55-kD glycoprotein composed of 362 amino acids in the rat

which strictly mediates sodium-dependent bile salt transport

favoring taurine-conjugated, trihydroxy-bile acids [9] This

protein is exclusively expressed in the hepatocyte at the soidal domain throughout the hepatic acinus Like other so-dium cotransporters, the concentrative uptake of bile salts is thermodynamically favored by coupling to a signifi cant out-side-to-inside sodium gradient maintained by the activity of

sinu-Na+, K+ATPase, assuring efficient uptake of bile salts at the sinusoidal surface Two sodium ions are transported for each molecule of bile salt Activity of the ntcp transporter is regu-lated by both its levels of expression at the sinusoidal surface and by gene transcription, which is an active area of investi-gation [3,4,10] Increased cAMP rapidly increases the con-tent of ntcp at the sinusoidal membrane by mobilizing an intracellular pool of transporters, the translocation being dependent on microtubule and microfilament activity [11] The ntcp expression is rapidly lost in primary cultured hepa-tocytes and all tumor-derived liver cell lines, suggesting tight regulation of this transporter, but it has been identified in human hepatocellular carcinoma samples Elevated serum levels of bile salts are associated with decreased expression whereas increased expression of the transporter gene has been noted postpartum due to prolactin [12,13] Increased

Figure 22.1 Transport proteins of the basolateral (sinusoidal) and

canalicular surface of human hepatocytes Proteins involved in the

hepatocellular uptake of organic anions include: Na+-taurocholate

cotransporting polypeptide (NTCP) and microsomal epoxide hydrolase

(mEH) (secondary active unidirectional transporters) and organic

anion transporting polypeptides OATP1B1, OATP1B3, and OATP2B1

functioning as bidirectional antiporters Multidrug resistance proteins

(MRPs), bile salt export pump (BSEP), and multidrug resistant gene product (MDR) are unidirectional primary active transporters involved

in the efflux of specific substrates listed with their respective transporters The substrate for FIC 1 is unknown Na+,K+ATPase is responsible for maintaining the low sodium, high potassium within the cell BS−= bile salts, OA − = organic anions, PC = phosphatidylcholine, BDG = bilirubin diglucuronide, CHOL = cholesterol.

bile salt fl ux does not regulate its expression in the rat [14]

Teleologically, decreased expression of ntcp protects the liver

in cholestasis in the face of elevated serum bile salts by

reduc-ing hepatic accumulation and potential toxicity of increased

concentration of intrahepatic bile salts Human NTCP is a 349

amino acid protein, which shares 77% sequence identity

with the rat [15] Of note, NTCP shares 50% sequence

identi-ty with the sodium-dependent ileal bile acid transporter,

re-ferred to as apical sodium dependent bile acid transporter

(ASBT) which is expressed at the lumenal (apical) domain of

the enterocyte [16,17] ASBT has also been identified in the

apical domain of large cholangiocytes, where it participates

in cholehepatic shunting, and in renal tubules cells [18,19]

Microsomal epoxide hydrolase (mEH), a key enzyme in

de-toxifi cation of reactive epoxides expressed at both the plasma

membrane and endoplasmic reticulum in the rat, has also

been implicated as a sodium dependent bile acid transporter

as its expression in a cell line can confer sodium-dependent

bile salt uptake [20] mEH expression seems to favor

glycine-conjugated bile salts

Organic anion and sodium-independent bile salt

transport

In addition to sodium-dependent bile salt transport,

dihy-droxy-bile salts and unconjugated bile acids can enter

hepa-tocytes via a sodium-independent transport mechanism

mediated by members of the organic anion transporting

pep-tides (OATP) superfamily Members of this large transporter

family are characterized by 12 membrane-spanning

do-mains containing a distinctive, superfamily peptide

signa-ture [21] These transporters may share common substrates

or be highly selective and are located in diverse tissues,

in-cluding the blood–brain barrier, liver, lung, intestine, and

kidneys Members of subfamilies are now defined by their

se-quence homology with proteins in humans, designated as

OATPs with the corresponding SLCO gene symbol As a class,

OATPs function as organic anion exchangers promoting

up-take of organic anions by exchanging them with effl ux of

other anions such as bicarbonate and glutathione [22,23] In

human liver, three OATP family members are expressed on

the sinusoidal domain [3,21] OATP1B1 is only expressed in

hepatocytes and facilitates the transport of organic anions

bound to albumin, including bile salts and their conjugates

and bilirubin, into the liver The restricted site of expression

of OATP1B1 in hepatocytes, coupled with its known

poly-morphisms, are likely to have a major impact on the

metabo-lism of pharmaceutical agents OATP1B3 is also expressed

in hepatocytes and shares 80% sequence identity with

OATP1B1, but is expressed in multiple tissues as well as in

cancer cell lines In addition to organic anions, it can

also transport small peptides such as cholecystokinin 8,

which differentiates it from OATP1B1 OATP2B1 is the other

family member expressed in the liver, as well as in spleen,

placenta, and lungs It has a narrower range of substrates as

compared to OATP1B family members and favors transport

of sulfobromophthalein (SBP) and estrone sulfate lar cloning of other oatp/OATP family members reveals a complex pattern of substrate specificity and distinct organ distribution in different species, suggesting that these trans-porters are involved with organ-specific uptake of specificorganic anions [3,21]

Molecu-Transcellular movement

Little is known about the transcellular movement of the key constituents of bile [24,25] Intracellular binding proteins with high affinity for bile acids, organic anions, fatty acids, and phosphatidylcholine have been identified, but their physiological function is still speculative [2,26] These pro-teins are assumed to target their hydrophobic ligands to dif-ferent intracellular components of the cell, including their respective canalicular transporters Vesicular-mediated transport of bile acids has also been implicated, based on in-creased transcellular movement of bile acids in response to cAMP and enhanced phospholipid excretion in response to infused bile acids These effects may also be due to increased insertion of canalicular transporters into the membrane Detailed studies with fluorescent bile acids have failed to demonstrate evidence for vesicular transport in isolated hepatocytes although these modified bile acids may not accurately reflect processing of bile salts [27]

Canalicular excretion

Canalicular membrane transport is the rate-limiting step in the vectorial movement of biliary constituents from the sinu-soidal space into bile Excretion of biliary components occurs across a relative concentration gradient of 100- to 1000-fold excess, requiring an active transport process Prior biochem-ical characterization of transport activity in canalicular-enriched plasma membrane vesicles has recently been advanced by the molecular identifi cation of specific canalic-ular transporters Detailed analysis of these individual trans-porters, coupled with loss of activities in rare human cholestatic syndromes and genetically engineered mice, has revealed their role in normal biliary physiology To date, all the canalicular transporters involved with biliary excretion are members of the diverse ATP binding cassette (ABC) class

of membrane transporters, in which transport of substrate is dependent on ATP hydrolysis [28,29] These proteins share either a single or dual magnesium-dependent ATPase region contained within a six-membrane-spanning domain with substrate specificity dictated by other regions of the trans-porters Major subclasses within this super gene family whose members play essential roles in biliary excretion in-clude the multi drug resistant (MDR) proteins, also known as P-glycoproteins, and the multidrug resistant proteins (MRP) Besides these ABC transporters with two ATP binding and catalysis domains, transporters with a single nucleotide binding domain also exist and function as key effl ux pumps

Another key gene involved with bile formation is a recently

identified P type ATPase, referred to as FIC 1 (ATP8B1), which

is responsible for Byler’s disease and benign recurrent

intra-hepatic cholestasis (BRIC) [30,31] We will review these key

ATP-dependent transporters and their role in the excretion

of biliary components

Canalicular bile salt transporter

As bile salt transport is increased by ATP hydrolysis, ABC

transporters of unknown function were screened as

poten-tial bile salt transporters [32,33] Using this strategy, a gene

similar to P-glycoprotein, which is exclusively expressed in

the hepatocyte at the canalicular domain, was considered

and shown to mediate ATP-dependent taurocholate

port in recombinant expression studies [34] This

trans-porter, now referred to as bile salt excretory peptide (BSEP)

(ABCB11) is composed of 1321 amino acids and shares 70%

identity with the P-glycoprotein [35] The gene is located on

chromosome 2 q24 in humans, which is identical to a shared

chromosomal region identified in children with a rare

pro-gressive familial intrahepatic cholestasis (PFIC 2) further

confirming its essential role in biliary excretion of bile salts

(see discussion of familial disorders below) [36,37] Kinetic

features coupled with unique expression at the canalicular

domain of the hepatocytes and its absence in PFIC 2 indicates

that this is the major, if not sole, bile acid transporter in the

canaliculus

Regulation of BSEP transport activity occurs at both

the transcriptional and more importantly at the

post-translational level, in which its redistribution from the

sub-canalicular endosomal pool to the sub-canalicular domain can be

rapidly enhanced during high bile salt loads Traffi cking

be-tween these pools is regulated by second messengers

includ-ing cAMP and PI3 kinase products, which enhance insertion

into the membrane [38,39] This rapid regulation of

expres-sion at the canalicular membrane permits dynamic

modula-tion of transport activity coupled to fl ux of bile salts by

altering the relative density of transporter

Canalicular phospholipid flippase

In addition to bile salts, phospholipids, such as

phosphatidyl-choline, and cholesterol are signifi cant components of bile

Elimination of murine Mdr2, which corresponds to MDR3

(ABCB4) in humans, by gene knock-out technology lead to

almost complete elimination of phosphatidylcholine in the

bile with a progressive cholangitis in these animals [26,40]

This ABC transporter is postulated to function as a “flippase”

transferring the energetically unfavorable movement of

phosphatidylcholine from the inner leaflet to the outer

leaf-let of the canalicular membrane Once at the outer leaflet,

phosphatidylcholine is extracted from the membrane by the

high concentration of bile salts present in the canalicular

space Inability to translocate the phospholipid to the outer

leaflet in the Mdr2 knock-out mice leads to bile salts freely

in-teracting with the luminal membrane of ductules, causing a detergent-induced cholangiopathy [41] In these Mdr2-deficient mice, progressive injury due to lack of buffering of bile salts leads to a progressive cholangiopathy and eventual hepatocellular tumor formation The human counterpart (PFIC 3) is discussed below

Cholesterol transport

Characterization of the molecular basis for sitosterolemia, an autosomal recessive disorder characterized by the accumula-tion of both plant-derived (primarily sitosterol) and animal-derived (cholesterol) sterols in plasma and tissues, lead to the identifi cation of two ABC transporters responsible for cho-lesterol effl ux [42] These ABC transporters are members of the ABCG subfamily, which are so-called “half-transport-ers” composed of a single, six-membrane-spanning region

with one ATPase domain ABCG5 (ABCG5) and ABCG8 (ABCG8) heterodimerize to form a functional pump for ex-

cretion of cholesterol and plant sterols They are coexpressed

in the canalicular domain and their overexpression in mice is associated with increasing canalicular excretion of choles-terol Like MDR3, these transporters are presumed to pro-mote movement of cholesterol from the inner to the outerleaflet of the canalicular domain, thereby functioning as a flippase

Canalicular organic anion transport

A multispecific organic anion transporter whose substrates include conjugated bilirubin, originally referred to as cMOAT and now as MRP2, was initially characterized in enriched canalicular membrane preparations whose activity was en-hanced by ATP hydrolysis [43] In humans, Dubin–Johnson syndrome is associated with persistent conjugated hyperbili-rubinemia and retention of hepatic pigment without other manifestations of cholestatic liver injury, suggesting specific

loss of MRP2 (ABCC2) These patients have a classical SBP

retention pattern in which a delayed regurgitation of conjugates in the serum occurs after initial clearance, indi-cating a deficiency in biliary effl ux Rodent models of this disease also exist, allowing for detailed analysis of bile com-position [44] This 1545 amino acid protein is expressed pre-dominantly in the apical domain of hepatocytes as well as proximal tubules of the kidney and duodenum and is one of eight MRP family members cloned to date [28] A peculiar feature of the animal models (TR−and EHBR rats) is the lack

SBP-of biliary GSH, indicating that mrp2 is essential for hepatic GSH excretion, a major contributor of bile-salt-independent bile flow [23]

Other members of the MRP family are also implicated in

biliary excretion of organic anions [28,29] MRP1 (ABCC1) is

minimally expressed in the liver but may be upregulated during cholestasis at the basolateral domain mrp3, which is located at the basolateral surface of hepatocytes, is upregu-lated in animals lacking mrp2 suggesting an alternative

pathway for elimination of organic anions across the

basolat-eral surface [46] MRP3 (ABCC3) is upregulated in patients

with primary biliary cirrhosis (PBC) as well, suggesting a

compensatory pathway in response to chronic cholestasis

[6] MRP3 is able to transport bile acids and their sulfated

and glucuronidated conjugates, which provides another

elimination route for retained bile salts in cholestatic

disor-ders Also, MRP3 is expressed on the basolateral aspect of

human cholangiocytes where it might participate in

chole-hepatic shunting and provide another means for the

elimina-tion of bile salts in cholestatic condielimina-tions In addielimina-tion to the

liver, MRP3 is extensively expressed in the small intestine

and kidney as well as the adrenal cortex MRP4 (ABCC4) and

MRP5 (ABCC5) have low levels of expression in the liver with

MRP4 being predominately expressed in the kidney and

prostate, and MRP5 expressed ubiquitously with greatest

levels in the brain and skeletal muscle Both these

transport-ers lack the amino terminal membrane-spanning domain

and thus are smaller then other MRP family members They

can both transport cAMP and cGMP nucleotides, which

dif-ferentiates them from other family members, and MRP4

preferentially transports conjugated steroids and estrogen

17β-glucuronide In the presence of ATP and GSH,

monoan-ionic bile salts are substrates for MRP4-mediated transport

across membranes, providing another route for effl ux of bile

salts out of hepatocytes during cholestasis MRP 6 is well

ex-pressed in the liver and has been localized to the basolateral

membrane and therefore may mediate the effl ux of organic

anions from the liver into the sinusoidal space during normal

and cholestatic conditions The substrates of MRP 6 have not

been characterized Surprisingly, mutations in MRP6

(ABCC6) are associated with pseudoxanthoma elasticum, a

disorder of connective tissues [28] Differential regulation of

these family members during cholestasis, with reduction of

apical MRP2 expression and induction of other MRPs

loca-ted at the basolateral membrane, provide alternative routes

for the effl ux of biliary-excreted compounds from

hepato-cytes into blood, thereby protecting hepatohepato-cytes from toxic

accumulation and allowing these substances to be excreted

by the kidney

FIC-1 P type aminophospholipid transporter

Rare disorders of progressive familial intrahepatic

cholesta-sis (PFIC-1) provide a unique opportunity to identify genes

associated with bile formation by searching for shared

chro-mosomal regions within selected affected populations

Link-age analysis of Byler’s disease in an Amish population and

benign recurrent intrahepatic cholestasis (BRIC-1) in a small

Dutch fi shing village revealed that these disorders share

the same chromosomal region at 18q21–22 [36]

Subse-quent identifi cation of the gene for FIC 1, now referred to

as ATP8B1, at this locus and discovery of mutations within

it in both clinical syndromes reveal that both diseases are

due to varying degrees of loss of activity of the FIC 1 protein [30] This protein shares amino acid sequence identity with

an aminophospholipid P-type ATPase, which mediates transfer of aminophospholipids from the outer to the inner leaflet of membranes, similar to the “flippase” activity of MDR3 The protein is expressed in the small intestine and pancreas with lower expression in the liver, including cholangiocytes and the canalicular domain of the hepato-cyte The mechanism by which the defect in this gene leads

to impairment of bile formation is unknown Recently, it has been shown that absence of FIC1 in ileal enterocytes leads to increased expression of ASBT and, consequently, in-creased bile acid absorption, which may overload the liver’s ability to maintain bile formation due to other abnormalities induced by this mutation Indeed, partial biliary diversion can ameliorate the clinical symptoms in some of these patients

Regulation of bile acid synthesis, transport, and metabolism by nuclear transcription factors

Like other steroid hormones, nuclear receptors play a key role

in regulating the de novo synthesis of primary bile acids from cholesterol, expression of bile salts, and organic anion trans-porters in both hepatocytes and enterocytes, and in the re-sponse of the liver to cholestatic injury Beside their essential roles in intestinal fat absorption and bile formation, synthe-sis of the primary bile acids has a major impact on global lipid metabolism and fatty acid synthesis in the liver We will pro-vide an overview of the current status of those transcription factors that regulate these diverse processes, which are largely based on studies performed in knock-out mice These receptors are listed in Table 22.2 and depicted in Fig 22.2 All these nuclear receptors share typical, modular protein struc-tures, which are listed from their amino (N) to carboxyl (C) terminal ends: A/B – contains the N terminal ligand inde-pendent transcription activator domain (AF1), C – the DNA binding domain, D – a shared domain and hinge region, and

E – the ligand-binding domain with the ligand dependent AF2 activation domain (F) located at the C terminal end of the protein The AF regions harbor binding sites for coactiva-tors or corepressors, which modify gene expression by inter-acting with the transcriptional machinery Differences in the amino acid sequence of the binding domain define the unique substrate specificity for these transcription factors These transcriptions factors most often heterodimerize with mem-

bers of the RXR nuclear receptor family (NRB1, 2, 3), whose

ligands include derivatives of retinoic acid [42,47] The

abili-ty of these factors to function as master co-ordinators for both metabolism and transport of bile salts and organic anions makes them ideal targets for the development of selec-tive agonists guided by the structural features of their

Table 22.2 Primary and secondary nuclear receptors regulating bile acid synthesis and response to cholestatic injury.

partner

Farsenoid X receptor FXR NR1H4 RXR Chenodeoxycholate > Induction of ABCB11, ABCC4, OATP1B3

Liver receptor homolog-1 LRH-1 NR5A2 none None Induction of CYP7A1, CYP8B1, ABCC3

Small heterodimer partner SHP NROB2 LRH-1 None Blocks LRH-1 dependent gene expression Pregnane X receptor PXR NR1I2 RXR Rifampin, natural as Repression of CYP7A1, CYP8B1, induction of

well as synthetic CYP3A11, SULT1A2, ABCC2, SLCO1B1

Constitutive androstane CAR NR1I3 RXR Required for Induction of ABCC3, ABCC2, CYP2B, SULT2A1

TCPOBOP, 1, 4, bis [2-(3,5 dichloropyridyloxy)] benzene (Summarized from [3,42,47,50–52,95,96].)

Figure 22.2 Primary and secondary nuclear receptor for bile acids in

hepatocytes Representative genes regulated by the primary bile acid

receptor, FXR, and the secondary receptors, PXR and FXR, are illustrated

When bound to bile acids, FXR directly induces expression of the gene

involved in canalicular bile salt transport, ABCB11, which encodes BSEP,

and canalicular organic anion transporter, ABCC2, which encodes MRP2

By induction of SHP, FXR can down regulate the first steps of primary

bile acid synthesis in both the neutral pathway, catalyzed by the gene

product of CYP7A1, and the acidic pathway, catalyzed by the gene

product of CYP8B1 SHP acts as a dominant negative receptor blocking

the transcriptional activity of LRH-1, a potent inducer of these two

genes The secondary bile acid receptors, PXR and CAR, have a lower binding affinity for bile acids as compared to FXR In addition, cholestatic bile acids, such as lithocholate and deoxycholate, as well as xenobiotics, are potent ligands for these transcription factors PXR and CAR induce expression of genes that encode efflux pumps, such as

ABCC2 (MRP2) and ABCC3 (MDR3) as well as the gene that encodes a

bile acid sulfotransferase, SULT2A1 Each nuclear receptor can also

induce a subset of specific genes When bound to its ligand, PXR induces

CYP3A family members, which can hydroxylate cholestatic bile acids

CAR ligands can activate expression of CYP2B family members while inducing expression of SULT2A1 and ABCC3.

ligand-binding domains In the near future, it is anticipated

that highly selective ligands for these factors will be used to

enhance cellular defense against cholestatic injury and

ame-liorate clinical symptoms The major nuclear receptors are

divided into the primary bile acid receptor, the farsenoid X

receptor (FXR), and secondary bile salts receptors, pregnane

X receptor (PXR) and the constitutive androstane receptor

(CAR), which are induced by xenobiotics as well as

choles-tatic bile acids

Farsenoid X receptor (FXR) (NR1H4)

In 1999, the primary bile acids, chenodeoxycholate and

cho-late, were found to be the endogenous ligands for the

previ-ously identified FXR receptor [42,47] FXR is responsible for

mediating bile-acid-induced down regulation of its own

synthesis, by indirectly reducing the expression of CYP 7A1,

the rate-limiting enzyme for the synthesis of primary

bile acids in the neutral pathway [42,47] FXR induces the

expression of SHP (NR0B2), a transcription factor that lacks a

DNA-binding domain, and binds with LRH-1 (NR5A2), a

potent promoter activator of the CYP 7A1 gene In the

absence of bile acids, LRH-1 potentiates the effects of LXRα

(NR1H3) thereby up regulating expression of CYP7A1 In

humans, chenodeoxycholate is the most potent natural

ligand while in mice, cholate is, and this may account for the

species-specific effects of FXR The reader is referred to Ory

and Edwards et al [42,47] for an in-depth review of how FXR

and these other nuclear receptors regulate global lipid

metabolism

In addition to down regulating CYP7A1, FXR bound with

bile acids increases expression of BSEP, which encodes the

major canalicular transporter of bile salts as well as enzymes

responsible for conjugating primary bile acids with taurine

or glycine Induction of these enzymes promotes formation

of bile salts from primary bile acids and ensures the

availabil-ity of canalicular transporters required for their

enterohe-patic circulation MDR3 is also up regulated, permitting the

co-ordinated excretion of bile salts and phospholipids into

bile FXR can also induce expression of MRP2, which

trans-ports bilirubin diglucuronides and other organic anions

FXR also regulates expression of the sodium-dependent bile

salt transporter on the ileal apical domain, ASBT, and the

in-tracellular bile acid binder, the intestinal fatty acid binding

protein (I-FABP) As FXR regulates expression of these bile

salts transporters, increased FXR signaling in the hepatocyte

is predicted to be protective against cholestatic injury by

pro-moting bile salt excretion The bile acid, ursodeoxycholate, is

routinely used for treatment of primary biliary cirrhosis, but

it is a poor FXR activator However, GW4064, a potent FXR

agonist, has been shown to decrease injury in a bile duct

li-gated model of cholestasis and to reduce the incidence of

gall-stone formation in mice [48,49] FXR agonists therefore

constitute a promising new class of agents for the medical

treatment of chronic, cholestatic conditions

Pregnane X receptor (PXR, NR1I2) and constitutive androgen receptor (CAR, NR1I3)

Beside FXR, studies in knock-out mice have demonstrated the importance of two well-recognized nuclear receptors, PXR and CAR, that enhance the metabolism of xenobiotics

by induction of cytochrome P450 (CYP) family members [3,50,51] PXR responds to a wide number of lipophilic com-pounds, including rifampin, due to its large ligand-binding

domain, and is responsible for induction of the CYP3A gene

family This family of enzymes catalyzes a monoxygenase tivity for diverse substrates, which is often the first step in the conversion of lipophilic compounds to more polar derivates

ac-In humans, the CYP3A gene family is responsible for

metabo-lizing approximately half of the prescribed drugs as well as endogenous and exogenous compounds Thus, PXR is an important factor in hepatic drug metabolism Recently, it has been shown that secondary, and especially cholestatic, bile salts, such as lithocholate, are potent PXR agonists, link-ing bile acid metabolism to their previous, well-established roles in xenobiotic metabolism CYP3A4 is able to hydroxyl-ate the cholestatic bile acid, lithocholate, at the 6 position, generating a more hydrophilic and less cholestatic bile acid Ursodeoxycholate is another PXR ligand In addition to the

CYP3A family, PXR inhibits CYP7A1 expression by a non-SHP

dependent pathway, demonstrating multiple sites for

regula-tion of the CYP7A1 gene Besides CYP3A members, PXR also induces expression of a sulfotransferase (SULT2A1), which

catalyzes sulfation of cholestatic bile salts and thus facilitates their renal excretion Members of the ABC transporter family, including MRP2 and MRP3, are also induced, allowing for the effl ux of conjugated bile acids out of hepato-cytes and into the vascular compartment for their eventual excretion in urine The effectiveness of rifampin to treat pruritus is likely due to induction of these PXR-dependent pathways

Besides PXR, CAR also participates in the metabolism and transport of cholestatic bile salts CAR’s mechanism of action differs from PXR and FXR, which are typically located in the nucleus, and activated by binding to their respective ligands

In contrast, CAR is activated in the absence of ligand and mally resides in the cytosol Some ligands directly bind to CAR in the cytosol whereas others, such as phenobarbital, lead to exposure of its nuclear localization domain causing it

nor-to migrate innor-to the nucleus where it binds at xenobiotic sponsive elements Although some of its ligands overlap with those that bind to PXR and both receptors can induce similar

re-genes, only CAR is able to selectively induce CYP2B family

members Studies in knock-out mice have demonstrated ferences in response to lithocholate with CAR inducing

dif-Cyp3a11 and AbcC3 whereas PXR induces Slco1B3, the

sinusoi-dal transporter, which is thought to enhance uptake of lestatic bile acids from the circulation [52] Sulfotransferases are also induced by CAR and catalyze production of sulfated bile salts, which can be excreted in urine These receptors

cho-represent additional targets for future therapeutic

interven-tions to treat cholestatic disorders

Regulation of key hepatic transporters in

experimental cholestatic conditions

Animal models have been developed to mimic different types

of clinical cholestatic syndromes which can occur with

either sepsis, bile duct obstruction, or high estrogen levels as

found in pregnancy [6], although species-specific responses

have been noted

Effect of estrogens

High doses of circulating estrogens have been implicated

in both intrahepatic cholestasis of pregnancy and oral

contraceptive-associated cholestasis [53] As the

concentra-tion of the synthetic estrogen, ethinyl estradiol, has been

re-duced in current oral contraceptive pills, this is a now a rare

cause of drug-induced cholestatic liver injury However,

in-trahepatic cholestasis of pregnancy is likely to be due, in part,

to high circulating estrogens or their metabolites, such as the

estradiol 17β glucuronide, which is known to be cholestatic

Treatment with the synthetic estrogen, ethinyl estradiol, in

rats leads to decreased bile flow and reduced excretion of

or-ganic anions, including bile salts and bilirubin, in the absence

of any morphologic changes, referred to as bland cholestasis

Estrogen-induced cholestasis is complicated by multiple

effects on membrane fl uidity, expression and location of

key transporters, as well as decreased enzymatic activity of

the Na+,K+ ATPase, a key regulator of sodium-dependent

cotransport processes Increased permeability across tight

junctions has also been implicated in cholestasis [53]

Estrogens affect transport activity at both the sinusoidal

and canalicular domain Decreased ATP-dependent

trans-port of organic anions and bile acids occurs in canalicular

membranes from ethinyl estradiol-treated animals

De-creased mrp2 and bsep protein expression in rats without

altering mRNA levels were found after ethinyl estradiol

treatment, indicating a post-translational dysregulation

[53,54] At the sinusoidal domain, decreased Na+,K+ATPase

activity was noted without a corresponding decrease in

mRNA or protein levels, indicating a functional change in

enzymatic activity which may be due to a secondary increase

in membrane fl uidity Decreased sodium-dependent

tauro-cholate uptake activity was associated with a progressive loss

of both ntcp protein and gene expression as well as Slco3a1

protein and gene expression [6,55] These effects observed in

rats may mirror similar mechanisms that contribute to

cholestasis of pregnancy in humans

Effect of lipopolysaccaride / endotoxin / cytokines

Cholestasis associated with sepsis is a well-described clinical

entity, which can be reproduced in animal models by

single-dose administration of endotoxin, the lipopolysaccaride

(LPS) component of the outer membrane of Gram-negative bacteria In rats treated with LPS, bile-salt-independent bile flow decreases with reduced expression of mrp2 in the cana-licular membrane which slowly recovers after 3 to 4 days Uptake of organic anions from the basolateral and canalicu-lar membrane fractions isolated from LPS-treated animals are reduced without altered transport kinetics, indicating a decrease in the total number of transporters [10,56] No changes in P-glycoprotein were found, indicating a selective down regulation of mrp2 canalicular transporter Decreased expression of ntcp protein and mRNA were also found, which

was mediated by a proximal regulatory element in the NTCP

gene

Regulation of different transporters in response to LPS jection is mediated by cytokine response Pretreatment of animals with corticosteroids prior to LPS prevents the reduc-tion in mrp2 expression by decreasing activation of NF-kB pathway, a key regulator for the induction of cytokines Treatment with anti-TNF antibody also abrogates the choles-tatic response Treatment with IL-1 or TNF-α can also mimic

in-effects of LPS on Ntcp gene expression whereas IL-6

treat-ment leads to reduced taurocholate uptake by reduction of

Na+,K+ ATPase activity without effecting Ntcp expression

[56] Thus, specific cytokines can contribute to cholestasis by modulating different components of the normal excretory pathway and provide targets for future treatments for choles-tatic disorders

Effect of bile duct obstruction

Animal models of acute bile duct obstruction mimic biliary tract obstruction due to stone, tumors, or stricture In rats, acute obstruction of the common bile duct leads to retention

of biliary content within the hepatocytes, further ing to cholestatic syndrome [14] Accumulation of second-ary, hydrophobic bile acid species can also reduce bile formation, increasing the cholestatic insult Absence of bile salts in the intestinal lumen can also promote translocation

contribut-of bacterial LPS NTCP gene expression is inversely correlated

with serum bilirubin levels [12], suggesting increased biliary content in the serum and liver can down regulate gene ex-pression Down regulation of sinusoidal transporters in this setting would protect the hepatocyte from further accumu-lation of toxic bile acid In primary biliary cirrhosis, OAT-P1B1 was decreased whereas BSEP and MRP2 was preserved and MDR3 and MRP2 were increased [57] There was a posi-tive correlation between the fractional percentage of ursode-oxycholate in the total hepatic bile salt pool and expression of OATP1B1 and MRP2

Clinical approaches to intrahepatic cholestasis and specific conditions

Cholestatic disease clinically means hepatobiliary disease predominantly caused by, and manifested by, impaired bile

secretion, usually without major liver destruction Thus,

acute and chronic viral hepatitis and alcoholic hepatitis and

cirrhosis commonly cause jaundice which undoubtedly

rep-resents failure of bile secretion but this occurs in the setting

of marked parenchymal cell death (elevated ALT) and

abnor-mal synthetic function (coagulopathy and low serum

albu-min) Cholestatic conditions typically are associated with

increased serum bile acids, markedly abnormal serum

alka-line phosphatase, and variable conjugated

hyperbilirubine-mia When bilirubin is normal to about 3 mg/dL, we refer to

this as anicteric cholestasis which is typical of PBC, primary

sclerosing cholangitis (PSC), chronic pancreatitis-associated

common bile duct stricture, and infiltrative diseases of the

liver Otherwise, when jaundice is present, it is referred to as

icteric cholestasis Since bile salts are the major solute that

determines osmotic bile secretion, retention and elevation of

bile salts in serum is a hallmark of cholestasis Selective

de-fects in bilirubin metabolism and transport, such as

MRP2-deficiency of Dubin–Johnson syndrome, cause

nonchole-static jaundice, which is not accompanied by increased serum

bile acids

Conceptually, cholestasis can occur because of mechanical

obstruction due to diseases of bile ducts: macroscopic

extrahepatic ducts (stones, stricture, intrinsic, or extrinsic

neoplasms) or destruction of microscopic intrahepatic ducts

(primary biliary cirrhosis, autoimmune cholangiopathy,

sarcoidosis, drug-induced vanishing duct syndrome)

Alter-natively, cholestasis can occur at the level of the

hepatocana-liculus as a result of a selective impairment in bile secretion,

which is the focus of the remainder of this chapter (see Table

22.3)

The work-up of cholestasis is covered in Chapter 4 Suffice

it to say that once extrahepatic duct obstruction has been

ex-cluded, one is working in the realm of intrahepatic

cholesta-sis In adults, a limited number of associated or causative conditions are pertinent, most of which will be obvious on clinical grounds: pregnancy, BRIC, sepsis, alcohol, postviral hepatitis, drugs, total parenteral nutrition (TPN), paraneo-plastic syndrome, and amyloidosis, along with the vanishing duct diseases (e.g PBC, sarcoid) From a diagnostic point of view, patients presenting with intrahepatic cholestasis should have blood cultures, viral hepatitis serology panel, serum mitochondrial antibody, and, if there is suspicion of infiltrative processes, imaging of the liver and abdomen It is important to remember that neoplasms that infiltrate the liver produce anicteric cholestasis but rarely cause jaundice

on the basis of infiltration

Genetic disorders presenting with cholestatic syndrome

Rare genetic disorders of progressive familial intrahepatic cholestasis clearly demonstrate the key function of these transporters and their clinical presentation due to loss of activity Subtle changes in functional activity of these key transporters, which leads to reduced but not absent activity,

or loss of a normal copy of the gene (heterozygous conditions) are being increasingly recognized as important risk factors for the development of cholestatic syndrome in response to clinical conditions such as sepsis, intrahepatic cholestatsis of pregnancy, or possibly drug-induced cholestatic liver injury Accumulation of genotyping information in these various conditions may ultimately identify genetic polymorphisms that could identify those patients at increased risk for devel-oping these cholestatic disorders

Progressive familial intrahepatic cholestatic (PFIC) dromes commonly present in early childhood with distinc-tive pathological and laboratory features and a normal biliary anatomy [2,58] Biliary atresia is the major cause of cholesta-sis in this age group whereas PFIC syndromes represent a dis-tinct minority If untreated, these patients may develop progressive liver injury requiring transplantation It is im-portant to identify genetic disorders in the bile acid synthetic pathway, in which toxic accumulation of normal precursors occur and are poorly transported by the canalicular trans-port system Molecular identifi cation of accumulated bile acid precursors by mass spectroscopy of the urine is highly effective for identifying these rare patients The PFICs have recently been classified according to the identity of the specific genes mutated in these disorders, as listed in Table 22.4 along with other disorders of the excretory pathway It

syn-is now increasingly recognized that adults presenting with benign, recurrent intrahepatic cholestasis (BRIC) carry mu-tations in the same transporters with less severe functional consequences

PFIC-1: Byler’s syndrome / disease

Byler’s disease, initially observed in descendants of the large Amish family of Jacob Byler, is characterized by recurrent

Table 22.3 Intrahepatic cholestasis, exclusive of intrahepatic duct

disease: PBC, sarcoid, Caroli’s disease, PSC, autoimmune

Alcoholic fatty liver

Total parenteral nutrition

Lymphoma and paraneoplastic syndrome

Amyloidosis

Drugs

episodes of jaundice which become persistent, with severe

pruritus along with elevated serum and reduced biliary

lev-els of bile salts [58,59] Patients with this disease who are not

direct descendants of Byler are referred to as having Byler’s

syndrome Patients have intermittent diarrhea and failure to

develop due to fat malabsorption A unique feature of these

patients is the normal level of serum γ glutamyl

transpepti-dase, which differentiates this from other progressive

choles-tatic syndromes in children [58] In general, patients are

compound heterozygotes with missense, chromosomal

dele-tions, inserdele-tions, or splice site mutations Patients with the

same mutations may have different presentations or can be

asymptomatic, indicating that other genes or environmental

factors may modify clinical presentation [60] These patients

may develop progressive liver disease within the first decade

of life and require liver transplantation In some patients,

partial external biliary diversion results in a substantial

im-provement in symptoms and regression of hepatic injury,

suggesting that cholestatic liver injury is a consequence of

cholestatic factors produced in the intestine Loss of FXR

activity in the intestine of PFIC11 patients has been observed

as well, which may contribute to cholestatic liver injury It

is speculated that loss of FIC 1 alters signaling leading to

decreased nuclear FXR As a consequence of decreased SHP

expression, ileal ASBT expression increases, leading to

in-creased bile acid absorption Furthermore, dein-creased FIC 1 in

the liver might decrease FXR, leading to decreased BSEP and

increased NTCP, but this remains to be shown

PFIC-2

PFIC 2 was originally identified in rare familial pediatric

cholestatic disorders which presented with similar serum

chemistry as Byler’s disease but affected individuals lacked

the commonly shared PFIC 1 chromosomal region [61]

These patients presented with a more aggressive cholestatic

syndrome soon after birth with rapidly developing liver

failure requiring transplantation and were unresponsive to

ursodeoxycholate therapy [62] Linkage analysis revealed

shared chromosomal region in afflicted patients at position

2q24, the same location as the BSEP gene [36,37]

Subse-quent studies in these patients confirmed mutations in

ABCB11 leading to absence of protein expression [63] The

absence of the characteristic elevation in the serum γ myl transpeptidase in severe cholestasis and minimal ductal injury observed in liver biopsies indicates that bile salts with-

gluta-in the biliary tree are essential for duct gluta-injury associated with increased serum γ glutamyl transpeptidase

PFIC-3

Patients who present with a similar pattern of cholestatic liver disease as PFIC 1 and 2 but with elevated serum γ gluta-myl transpeptidase were shown to have mutations in both al-

leles of the human ABCB4 gene which encodes MDR3, a

transporter with phospholipid flippase activity [58,64]

These patients, as in the corresponding Mdr2 knock-out mice,

have severe ductal injury due to lack of buffering of bile salts

by phospholipids [41] Histologically, these patients have portal fibrosis with ductular proliferation and infl ammatory infiltrate despite patency of the bile ducts Clinically, these patients present at later stages than PFIC 1 and 2 with more symptoms related to cirrhosis, such as portal hypertension Interestingly, some cases of cholestasis of pregnancy have been observed in heterozygote carriers of these same MDR3 mutations (see below)

Benign recurrent intrahepatic cholestasis (BRIC)

The diagnosis of BRIC should always be considered in tients with recurrent episodes of cholestasis in the absence ofbile duct obstruction, or infiltrative or chronic liver disease Genetic analysis of the transporters responsible for the PFIC syndromes have now revealed the molecular mechanism for

pa-some of these disorders Initially, mutations in the FIC1 gene, now referred to as ATP8B1, were found in both patients with

BRIC and PFIC-1 The severity of cholestatic liver caused by

mutations in ATP8B1 disease refl ected the relative activity of

the FIC 1 transporter [30,60] Both PFIC-1 and this form of BRIC, now referred to as BRIC-1, are inherited as autosomal recessive diseases BRIC-1 initially presents in childhood or during early adolescence and reoccurs throughout adult-hood During cholestatic episodes, patients present with a 1- to 2-week prodrome of pruritus and anorexia before onset

of jaundice, which can last from 1 to 3 months and ously resolves Viral illness, pregnancy, or winter have been temporally associated with the onset of cholestatic episodes but often no precipitating event can be identified Diets rich

spontane-in fatty acids and oral contraceptives have also been plicated as precipitating factors In between episodes, pa-tients have a contracted bile acid pool, which is enriched with conjugates of secondary bile acids[65] Recently, 20 individ-uals with BRIC symptoms but no identified mutations in

im-ATP8B1 were evaluated for mutation in the ABCB11 gene,

which encodes for BSEP [66] Homozygous or compound

Table 22.4 Hereditary disorders of liver transporters causing jaundice.

PFIC 1 (Byler’s disease/ FIC1 ATP8B1 18q21–22

syndrome)

PFIC 2 BSEP ABCB11 2q24

PFIC 3 MDR3 ABCB4 7q21

BRIC-1 FIC 1 ATP8B1 18q21–22

BRIC-2 BSEP ABCB11 7q21

Dubin–Johnson MRP2 ABCC2 10q23–24

syndrome

heterozygote mutations were detected in 10 of these 20

pa-tients and in one patient, only a single mutation was found

These patients, now referred to as BRIC-2, differed from those

with mutations in ATP8B1 (BRIC-1) by their absence of

wa-tery diarrhea, pancreatitis, or hearing loss Some patients

developed permanent cholestasis with age and liver biopsies

typically demonstrated histologic features of cholestasis

without fibrosis During cholestatic episodes, serum bile salts

were elevated In two women, cholestasis developed during

pregnancy Treatment with rifampin, cholestryamine, or

ur-sodeoxycholate may hasten resolution of cholestatic episode

but not prevent their occurrences [62] Like 1 and

PFIC-2 patients, these BRIC-1 and BRIC-PFIC-2 patients have elevation

of serum alkaline phosphatase levels with normal serum γ

glutamyl transpeptidase, which differentiates BRIC from

other cholestatic syndromes in adults In the future, genetic

testing may be used to identify the etiology and confirm the

diagnosis of BRIC

Total parenteral nutrition associated

cholestatic syndromes

Long-term total parenteral nutrition (TPN), especially in

ne-onates, is associated with a nonobstructive cholestatic

syn-drome and can lead to liver failure Correlation between

incidence of cholestasis and TPN in adults is complicated by

underlying conditions and the different indications for TPN

support [67,68] In adults, cholestasis is the predominant

he-patic abnormality, which occurs after 3 weeks or longer of

therapy and presents with increased alkaline phosphatase,

occasionally associated with serum bilirubin elevation Liver

biopsy typically reveals canalicular bile plugs in periportal or

perivenous zones in combination with mild portal triaditis

The cholestasis is attributed to multiple etiological factors

ei-ther directly related to the TPN solution or the underlying

clinical conditions Increased uptake of amino acids by the

liver in combination with decreased excretion of normal

bili-ary constituents and steatosis, secondbili-ary to the high

carbo-hydrate concentration of the TPN solution, are risk factors

considered to be responsible for cholestasis Reduced mdr2

expression preceding liver injury was noted in mice treated

with TPN, suggesting loss of normal canalicular function as a

cause for this cholestatic condition [69] Lack of oral intake

with loss of the normal enterohepatic circulation of bile salts

is associated with a 10-fold increase in TPN-associated

cho-lestasis In these patients, greater production of cholestatic

secondary bile acids is another factor Patients with ileal

dys-function, such as with Crohn’s disease, are also at greater

risk, further indicating that the lack of a normal

enterohepat-ic circulation is an important factor Intercurrent sepsis is

another risk factor and by itself may cause cholestasis as

previously described Cessation of TPN leads to resolution of

the cholestasis in most cases Alternatively, reduction in

car-bohydrate content of TPN, treatment with oral antibiotics,

and small enteral feeding to promote the enterohepatic

cir-culation can be tried while closely monitoring cholestatic markers In anecdotal case reports, UDCA treatment im-proved TPN-associated cholestasis It is important to recog-nize that these patients are also at increased risk for formation

of biliary sludge and gallstone formation leading to titis and/or cholangitis which can present with right upper quadrant pain and fever associated with increased alkaline phosphatase and jaundice Prompt recognition and imaging

cholecys-of the liver and biliary system is required to make the sis and initiate treatment

diagno-Cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy (ICP) is a rare disease

of unknown cause which may be recurrent in 40 to 60% of cases [70] Patients typically present in the second half of their pregnancy, initially with pruritus occasionally accom-panied by jaundice which rapidly resolves in the postpartum period Increased serum bile acids, which are predominately conjugated cholic acid, bilirubin, and moderately increased alkaline phosphatase and γ glutamyl transpeptidase are found No long-term sequelae have been noted for the mother but the fetus is at increased risk of premature delivery, fetal distress, and perinatal mortality In a study from Sweden, fetal complications were only identifi ed in those mothers whose serum bile salts were greater then 40 µmol/L [71], leading the authors to recommend that serum bile salts should be routinely monitored in patients with ICP Treat-ment with UDCA in clinical studies was associated with im-proved fetal outcome [72] These patients are also at risk for oral contraceptive-induced jaundice Etiology for the disease

is unknown but increased incidence of ICP in multiparous births which have higher estrogen levels suggest a possible defect in estrogen metabolism, leading to an increased pro-duction of the cholestatic estradiol 17β-glucuronide, as a po-tential mechanism for this disorder Widely different rates of ICP are noted in different countries, with Chile and Sweden having the highest rates, suggesting genetic factors are clearly important Recently, the incidence of ICP has de-creased in both these countries In anecdotal cases, muta-

tions in ABCC3 have been found in patients with ICP In one

patient presenting with recurrent cholestatic episodes, ICP

and eventually PBC, a novel mutation in ABCC3 encoding for

MDR3 was found [73] Linkage analysis in a nonrelated group of Finnish women with ICP revealed an association between ICP and a single nucleotide polymorphism in

ABCB11, which encodes for BSEP [74] However, another

study from Finland found no common regions in either the

ABCB11, ATP8B1, or ABCC3 genes in these women, suggesting

a genetically diverse group of disorders

Viral hepatitis and cholestastic liver disease

Jaundice with acute or chronic viral hepatitis is caused by loss of parenchymal function due to hepatocellular necrosis and/or fibrosis Rarely, viral hepatitis may present with cho-

lestasis as the predominant clinical feature and is most

fre-quently found in adults with hepatitis A viral (HAV) infection

[75] Unlike children, in whom HAV is often a subclinical

and anicteric illness, adults can present with severe

choles-tatic syndrome mimicking chronic bile duct obstruction

Pa-tients complain of pruritus, fever, diarrhea, and weight loss

and symptoms may last 1 to 4 months A relapsing course can

occur in which an initial apparent improvement, lasting 3 to

5 weeks with normal serum chemistries, is followed by

re-currence of cholestatic serum markers and symptoms for an

additional 4 to 6 weeks Liver biopsy reveals intraductal

cholestasis and portal tract infl ammation associated with

paucity of bile ducts

In anecdotal case reports of HAV, treatment with oral

pred-nisone decreased jaundice and improved cholestatic

symp-toms This treatment is not recommended for other viral

hepatitides HAV in adults should always be considered in the

differential diagnosis of intrahepatic cholestasis Rarely,

other causes of acute and chronic viral hepatitis can present

with predominant cholestatic features Case reports of

cho-lestasis in chronic hepatitis C virus infection, acute

cytomeg-alovirus in immunocompetent individuals, and Epstein–Barr

virus infection have also been reported [76,77]

Cholestatic syndrome with ethanol

Occasionally, patients with alcoholic liver disease may

pres-ent with a cholestatic syndrome in the absence of cirrhosis or

alcoholic hepatitis Initial reports associated alcoholic

intra-hepatic cholestasis with marked steatosis [78,79] Patients

present with malaise, anorexia, and hepatomegaly with a

cholestatic pattern of serum liver tests Compared to patients

presenting with alcoholic hepatitis, these patients tended to

have poorer nutritional status, greater alcohol consumption,

and a worse prognosis Treatment consists of nutritional

sup-plementation and supportive care after imaging of the liver to

eliminate biliary obstruction or infiltrative disease Chief

histologic features of alcoholic cholestatic liver are

macrove-sicular fat with some microvescicular fat in centrolobular

he-patocytes, portal tracts infiltrated with infl ammatory cells

and proliferating bile ducts Fibrosis extending from the

por-tal tract into the liver acinus is routinely found without

Mal-lory bodies and minimal focal necrosis In one large Veteran’s

Administration cooperative study, a retrospective review of

patients with cholestatic features on liver biopsy was

as-sociated with increased serum alkaline phosphatase and

signifi cantly decreased survival compared to those without

cholestatic features [80]

Some patients with a cholestatic presentation have

pre-dominantly microvesicular steatosis (alcoholic foamy fatty

disease) They may have jaundice and variable, sometimes

markedly elevated, serum alkaline phosphatase and

aspar-tate aminotansferase (AST) (up to 1000) Liver biopsies

re-veal cholestasis, predominant centrilobular microvesicular

fat, and little infl ammation or necrosis This picture has been

associated with deletions of mitochondrial DNA Although occasionally fatal, most patients improve rapidly after alco-hol is withdrawn

Sepsis-associated cholestatic syndromes

Cholestatic liver disease accompanying systemic sepsis is a well-recognized clinical syndrome and is hypothesized to be due to increased cytokine production The development of jaundice with sepsis is a poor prognostic indicator due to the gravity of the underlying infection Cholestatic liver disease can be caused by Gram-positive and Gram-negative bacterial and fungal infections Sepsis is the predominant clinical fea-ture in these patients, who do not complain of right upper quadrant pain or pruritus Serum alkaline phosphatase is routinely elevated (but only mildly) with conjugated hyper-bilirubinemia occurring to a variable degree Occasionally, 10-fold or greater increased serum alkaline phosphatase in the absence of jaundice has been reported [81] In one study, cholestasis and increasing hyperbilirubinemia were associ-ated with multisystem failure and increased mortality [82]

No specific therapy for the cholestasis is necessary in these patients, after excluding the liver or biliary system as the site

of infection, and cholestasis resolves with treatment of the underlying infection Jaundice without increased alkaline phosphatase may be a heralding sign for impending sepsis [83] Liver biopsy in severe sepsis-associated cholestasis re-veals inspissated bile with dilated and proliferating portal and periportal bile ductules Cholestasis has also been re-ported in toxic shock syndrome with increase serum bile acids, mild transaminases, and hypoalbuminemia

Paraneoplastic cholestasis

Cytokines play a key role in mediating cholestasis associated with sepsis Increased cytokine production associated with neoplastic diseases is now presumed to be responsible for rare cholestatic disorders associated with malignancy in which there is no bile duct obstruction or infiltration of the liver pa-renchyma Anecdotal case reports in Hodgkin’s disease and other lymphomas have noted an increase in alkaline phos-phatase and bilirubin in the absence of infiltrative disease [84] Stauffer’s syndrome, a paraneoplastic syndrome asso-ciated with renal cell carcinoma, which presents with cho-lestasis, fever, increased acute phase reactants and anemia, is associated with increased serum IL-6 levels Experimental treatment with anti- IL-6 antiserum in these patients led to a temporary decrease in alkaline phosphatase, which returned

to their elevated levels after cessation of treatment indicating that IL-6 was responsible for cholestasis [85] Patients treated with IL-2 were also found to develop reversible cholestasis, as evidence by increased serum bile acids, bilirubin, and alka-line phosphatase which returned to normal after completion

of treatment [86] Prostate cancer may also present with a cholestatic pattern of liver involvement without evidence for biliary obstruction, which resolves with implementation of

antiandrogen therapy [87] It is likely that excess cytokines

will also be responsible for other paraneoplastic cholestatic

syndromes

Amyloidosis

Although amyloid infiltrates are commonly found in the

liver, patients rarely present with jaundice Hepatomegaly

associated with amyloid has been attributed to the associated

congestive heart failure and not liver infiltration Rarely,

am-yloidosis may initially present as intrahepatic cholestasis

[88] Histologically, these patients present with

perisinusoi-dal deposition of amyloid paralleled by advanced

hepatocel-lular atrophy with varying degrees of bile stasis Patients with

amyloidosis exhibit a low serum gamma globulin in contrast

to the elevated serum globulin levels found in chronic liver

disease

Drug-induced cholestasis

A wide variety of drugs can cause cholestatic liver disease

Aside from the bland cholestasis (no infl ammation) seen

with androgens and estrogens, which probably occurs in

in-dividuals with a genetic predisposition to cholestasis,

drug-induced cholestasis tends to exhibit portal tract infl ammation

and bile duct injury along with variable parenchymal

in-fl ammation and necrosis/ apoptosis Indeed, many of the

drugs that induce clinical cholestasis with jaundice, pruritus,

and marked increased alkaline phosphatase also are

associ-ated with moderate to marked parenchymal destruction, as

reflected in elevated alanine aminotransferase (ALT) Thus,

the same drug in some individuals will produce

predomi-nantly cholestasis, in some mixed cholestasis/ hepatitis, and

in some predominantly hepatitis [89] Table 22.5 lists drugs

that either usually produce predominantly cholestasis or

most often produce cholestasis but with variable and

some-times predominant hepatitis The jaundice in this condition

tends to resolve very slowly after discontinuing the drug,

sometimes lasting for months and even evolving into a

van-ishing duct/ biliary cirrhosis picture (Table 22.6)

A very high proportion of the drugs that produce clinical

cholestatic disease seem to do so on an immunoallergic basis;

this is based on early onset, in the first few weeks of therapy,

and the concomitant appearance of fever, rash, eosinophilia,

and, in some instances, positive rechallenge In the case of

certain drugs, for example antibiotics such as erythromycins

or Augmentin®, following a course of 1 to 2 weeks, the

chole-static syndrome appears up to 3 to 4 weeks after

discontinu-ing the drug A recent analysis of HLA polymorphisms in a

large group of patients with drug-induced liver injury

identi-fi ed an association with cholestatic reactions, supporting the

predominantly allergic nature of this adverse phenomenon

with many drugs [90]

Whether or not features of allergy are present, the

patho-physiology is uncertain However, the target of many of these

immunological reactions appears, on circumstantial

evi-dence, to be the bile ductules, which are often associated with infl ammation and not infrequently with progressive de-struction (Fig 22.3) However, parenchymal infl ammationand cytokines may contribute by down regulating the vari-ous hepatic transporters, leading to impaired bile acid and organic anion secretion In theory, this would be more likely

to be clinically manifest in patients with a genetic tion, such as heterozygotes for defects in the transporters This might account for the fact that many of the drugs commonly induce mild anicteric liver test abnormalities but rarely cause overt liver disease In addition, BSEP-mediated

predisposi-Table 22.5 Some drugs that can lead to cholestasis (Reproduced from

Zimmerman [89], with permission from Lippincott Williams and Wilkins.)

Cholestatic injury Cholestatic or characteristic hepatocellular injury

Ajmaline Allopurinol Amoxicillin-clavulanate Antidepressants (Tricyclic, tetracyclic) Anabolic steroids Captopril

Benoxaprofen Carbamazepine Benzodiazepines Cimetidine Butyrophenones Clozapine Carbimazole Droxicam Cloxacillin Enalapril Cyproheptadine Fluconazole D-Propoxaphene Gold compounds Danazol Hydralazine Dicloxacillin Itraconazole Erythromycins Ketoconazole Flucloxacillin Naproxen Griseofulvin Nitrofurantoin Methimazole Phenylbutazone Oral contraceptives Phenytoin Penicillamine Piroxicam Phenothiazines Ranitidine Sulfonylureas (most) Sulfonamides Thiabendazole Sulfamethoxazole-trimethoprim Thioxanthines Sulindac

Ticlopidine Zidovudine Troleandomycin

Xenalamine

Table 22.6 Drugs incriminated in chronic cholestasis (Reproduced

from Zimmerman [89], with permission from Lippincott Williams and

Wilkins.)

Aceprometazine (with meprobamate) Erythromycins

Ajmaline and related drugs Estradiol

Figure 22.3 Pathogenesis of drug-induced

cholestasis Drugs can inhibit canalicular

BSEP (e.g cyclosporin A, rifampicin), be

converted to toxic metabolites which might

elicit an immune response directed at

hepatocytes or bile ducts (via secretion), or

an associated inflammatory response which

might lead to cytokine-mediated down

regulation of hepatocyte export pumps.

bile acid transport is competitively inhibited by cyclosporin

A [91–93], rifamycin SV, rifampicin, glibenclamide, tan, troglitazone, erythromycin, and estolate, whereas only cyclosporin among this group inhibits MRP2 [91] Except for mild cholestasis with cyclosporin, it is doubtful that the inhi-bition of BSEP by the other drugs causes clinical cholestasis Sulindac also inhibits canalicular bile acid transport [94] Interestingly ethinylestradiol-17β-glucuronide is secreted into bile by MRP2 and then transinhibits BSEP [91] Another intriguing possibility, with limited proof, is that toxic but sta-ble metabolites of certain drugs, that are secreted into bile, exert toxic effects on the bile duct system The best experi-mental support for this mechanism derives from studies of α-naphthylisothiocyanate (ANIT) ANIT forms a labile GSH adduct in hepatocytes After concentrative pumping into bile, the adduct dissociates in alkaline bile and ANIT, at high concentrations, attacks cholangiocytes Rats with mutation

bosen-in mrp2 do not pump the adduct bosen-into bile and are protected from ANIT-induced cholestatic injury

Questions

1 Which serum test can be used to differentiate the etiology of

progressive familial intrahepatic cholestasis?

2 Which of the following transport mechanisms are not

operational at the sinusoidal membrane of hepatocytes?

a inability to synthesize primary bile acids

b lack of active transport of bile acids

c extraction of lipids from the apical domain of hepatocytes and

cholangiocytes

d inability to secrete cholesterol into bile

e increased SHP expression

Suggested readings

Kullak-Ublick GA, Stieger B, Meier PJ Enterohepatic bile salt

trans-porters in normal physiology and liver disease Gastroenterology

2004;126:322–42.

Excellent review of the current understanding of bile formation,

fea-tures of hepatic transporters and their regulation in human and animal

models of cholestatic liver disease.

Jansen PL, Sturm E Genetic cholestasis, causes and consequences

for hepatobiliary transport Liver Int 2003;23:315–22.

Update and concise review of clinical features and the genetic

muta-tions in liver transporters responsible for progressive familial

intrahe-patic cholestatic syndromes in children.

3 Kullak-Ublick GA, Stieger B, Meier PJ Enterohepatic bile salt

transporters in normal physiology and liver disease

Gastroen-terology 2004;126:322–42.

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charac-terization, function, and regulation Physiol Rev 2003;83:

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12 Shneider BL, Fox VL, Schwarz KB, et al Hepatic basolateral dium-dependent-bile acid transporter expression in two un- usual cases of hypercholanemia and in extrahepatic biliary atresia Hepatology 1997;25:1176–83.

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