Cholestasis, Molecular Pathogenesis

[Trauner M, Meier PJ, Boyer JL. NEJM (1998) 339 1217-26]

Bile formation:

• Osmotic secretory process
• Driven by active concentration of bile salts in the bile canaliculi
• Transport mechanisms in the basolateral (sinusoidal) and apical (canalicular) surfaces of the hepatocytes. There are absolutely stacks of these on both membranes. Transport on the across the canalicular membrane side of the hepatocyte is the rate limiting step.
  • Sinusoidal membrane
    • Na/K ATPase maintains –35mV charge which drives the Na/H+ pump (protons out of the cell), HCO3-/Na+ (bicarbonate entry) and Na+ dependent uptake of conjugated bile salts or bile acids.
    • Bile salts are the most abundant solutes in bile
    • Transport from plasma into hepatocytes is mediated by the sodium taurocholate cotransporter.
    • Other non-conjugated bile salt (cholate) and lipophilic albimin-bound compounds are transported from plasma into hepatocytes via a sodium-independent transporter.
  • Canalicular membrane
    • Rate limiting step
    • Transport via ATP-dependent pumps (ATP-binding cassette family of membrane transporters)

E.g.: multidrug resistance-1 P-glycoprotein (MDR1) – mediates transport of bulky lipophilic cations (e.g.: anticancer drugs, cyclosporine A, etc).

• • • Physiological role – unclear, ?substrate

MDR3 – liver specific function cf: MDR1, it transports phosphatidylcholine from the inner to the outer leaflet of the canalicular membrane.

• • • Canalicular multispecific organic-anion transporter – a canalicular form of the multidrug resistance-associated protein (MDP2) – mediates transport of leukotriene C4, glutathione-S conjugates, glucuronides (bilirubin diglucuronide, estrodiol-17β- glucuronide) largely responsible for generation of bile flow independent of bile salts within the bile canaliculi.
    • Canalicular bile salt transporter – probably a member of the ATPase binding cassette family.

• Bile flow also affected by exocytosis of transcytotic and subcanalicular vesicles; activities of peptidases, nulceotidases, periodic contractions of bile canaliculi, bile ductule and hepatocyte ion channels, etc.
• Chloride is excreted by the chloride/bicarbonate anion exchanger and the cystic fibrosis transmembrane regulator (CFTR) (Cl- channel on the luminal surface of the bile duct epithelial cells).

Animal models of cholestasis:

• Endotoxin treated rats – sepsis-induced cholestasis
• Oral-contraceptive treated animals
• Ethinyl estradiol-treated rats – cholestasis of pregnancy
• Extrahepatic – ligation of common bile duct
• Many drugs cause cholestasis e.g.: cyclosporine A, chlorpromazine

Molecular mechanisms of cholestasis

Hepatocellular transporters

• Various inherited disorders of hepatocellular transport seen in humans that can result in reduction in bile flow and therefore cholestasis.

• Phospholipids within normal bile protect the bile ductule epithelial cells from the toxicity of bile salts by forming mixed micelles – there is a mutation in the MDR3 gene that causes marked decrease in PL transport into the bile  hence bile ductule damage and inflammation. Patients with primary biliary cirrhosis have normal MDR3 expression though…

• Dubin-Johnson disease – mutation in a MRP2  hyperbiliruninaemia but not cholestasis due to inappropriate excretion of endogenous conjugates (bilirubin digluconuride and coproporphyrin I). Seen in South Down sheep and Corridale sheep.

• Acquired forms of cholestasis may have effects on the basolateral or canalicular transport mechanisms e.g.: altered expression of the transport proteins. Molecular alterations in basolateral transport may contribute to the functional impairment of bile formation by diminishing the hepatocellular uptake of biliary constituents. Also may reduce build up of toxic substances (bile salts, etc) within hepatocytes. Promotors to these transporters have signal sequences transcription regulating factors that are responsive to bile salts, cytokines, etc. Indeed, endotoxin-induced cholestasis in rats is caused by inhibition of activity of hepatocyte nuclear factor 1 (and other cytokines) that decreases expression of transport proteins.

Organic-anion transporting polypeptide is upregulated in cholestatic disease – possibly helping to export toxic substances out of hepatocytes and into the bile canaliculi.

• As the transport across the canalicular membrane is the rate limiting step, it is one of the major causes of cholestasis – expression of the transport proteins across this membrane are downregulated in experimental cholestasis models (e.g. decreased expression of bile-salt export pump, multispecific organic-anion transporter).
• Bicarbonate exchangers also play a role in the decrease in canalicular and ductular bile flow (some patients have generalized syndromes that also affect saliva secretion as well).

Cholangiocyte transporters

• CFTR – on cholangiocytes and not on hepatocytes – mutations impair ductular secretion of chloride and water.

• Other defects:
• Most cholestatic liver disease causes profound hepatocyte cytoskeletal changes – disruption of microtubules, increases in intermediate filaments, accumulations of disorganized actin microfilaments in the pericanalicular domain. Result in loss of apical microvilli, decreased contractility of the canalicular membrane, increased permeability of intercellular tight junctions (latter results in regurgitation of bile into plasma, reduction of osmotic gradients in the bile canaliculi (the driving force for bile secretion)).
• Changes in the tight junction permeability is affected by the altered location and accumulation of the tight junction proteins (occludin and zonula occludens I).
• Also membrane components, transcytosis and canalicular exocytosis are also altered during cholestasis. Results in retention of apical transporters on the basolateral surface of hepatocytes and a delay in vesicle transport to the bile canaliculi. Accumulations of vesicles within the pericanalicular region of hepatocytes is characteristic of choleostatic liver injury.
• High concentrations of bile salts inhibit function of molecular motors (dynein and kinesin) that move the vesicles.
• Calcium signaling within and between hepatocytes decreases with choleostasis – changes in gap junctions so may change microperistalsis down the terminal canaliculi to the bile ducts in the portal triads (counter to direction of blood flow).
• cAMP mediated signaling in the hepatocyte is impaired due changes in G protein expression and localization. This together with changes in membrane composition and the detergent effects of the bile salts – contribute to decreased adenylate cyclase activity. Also therefore decrease effects of glucagons and VIP that normal stimulate bile secretion.