Molecular Physiopathology of Cholestasis
Cholestasis is the result of a diminution of biliary secretion to the intestine. Biliary secretion is carried out through transporters located in the hepatocytes and cholangiocytes (N Engl J Med 1998; 339: 1217-1227).
A cholestatic syndrome may overcome after a mechanical obstruction of the common bile duct or secondary to biliary tree diseases or hepatocyte diseases (genetic diseases, infectious, autoimmune, metabolic, neoplastic, or toxins).
The consecuences of this syndrome are related to biliary acid toxicity that accumulates in the liver and body, or to their absence in the digestive tube, interrupting the enterohepatic cycle.
Biliary Secretion
The bile produced by the hepatocyte inside the canalicular duct, enters the bile ducts, then to the larger bile ducts until it reaches the duodenum. The biliary canalicular duct is the smallest structure in the biliary tree, it’s wall is constituted by the heaptocyte’s canalicular membrane. On this membrane there are transport proteins that allow the transport of osmotically active substances against a concentration gradient in the hepatocyte towards the bile duct.
Bile formation by the hepatocytes is done by a difference in osmotic pressures, which appears from the bile acid concentration inside the bile ducts and from other substances such as gluthathion. Water and electrolyte movements occur esentially through a paracellular mechanism, through intercellular junctions.
Bile acids, phospholipids, and cholesterol, all associate inside the bile under the form of mixed mycelles. Maintenance of the hepatocytes’s polarity (canalicular membrane and basolateral membrane) is esential for biliary secretion.
Portal Flow Transport Inside the Hepatocyte
Bile acids converge in the sinusoidal pole of the hepatocyte by the portal blood. They are transported inside the hepatocyte against a concentration gradient.
Over the hepatocyte’s basolateral membrane a Na-K+ ATP as has a role in the maintenance of the transmembrane gradient of sodium ionside the cell. This pump and the potassium channels allow the maintenance of a transmembrane electric potential of -35 mV. This produces the necessary energy for sodium secretion and bile acid transport.
The bile acid transporter is Na+ taurocholate co-transporter popypetide (NTCP), which transports sodium dependent conjugated bile acids. Bile acids and other lipophylic substrates may be transported as well inside the hepatocyte by an independent transporting system not dependent on sodium called organic anion transporter polypeptide (OTAP) (Am J Physiol 1995; 269: G801-G812).
Intrahepatocyte Transport
Once inside the hepatocyte, the different osmotic substances join the canalicular pole. Contrary to the progress made in the knowledge of these dfferent mechanisms of canalicular transport, intracellular ransport is still unknown.
It seems that, contrary to what was thought years before, transport doesn’t occur by a vesicular mechanism, but by intracellular transport proteins. Different proteins have been identified which are still being studied (Semin Liver Dis 2000; 20: 273-292).
Canalicular Transport
Under physiological conditions, the limiting stage in biliary secretion is the canalicular transport of bile acids. The canalicular transporter of bile acids has been identified. This transport is stimulated by ATP. The protein has been called: bile salt export pump (BSEP), sister P-glycoprotein (SPGP), ATP binding cassette sub-family B11 (ABCB11).
At the canalicular membrane level, other transporters implicated in biliary secretion have been identified. They are also ATP depending transporters of the family of ATPases type P. Some of them such as mdr1, plays an important role in the canalicular elimination of many lipophylic molecules. Another is mdr3 which plays a role in the biliary secretion of phospholipids.
Vesicular Bile Formation
Canalicular bile doesn’t have the same composition as vesicular bile. Particularly, epithelial ductal cells or cholangiocytes posess transporters (CFTR and AE2) which facilitate chloride and bicarbonate secretion. Bile ducts secrete a bicarbonate rich solution under the influence of secretin. The gallbladder reabsorbs water and electrolytes, and concentrates bile to a concentration 10 times the normal.
Active Transports of the different molecules inside the biliary duct allow biliary secretion, but it is not efficacious if the hepatocyte is not intact. Particularly, cell polarity (separation of the sinusoidal and canalicular membrane of the hepatocyte), the cytoskeletal integrity, and the impermeability of the intercellular junctions and homeostasis of the calcium pool inside the hepatocyte must be preserved.
Complications of Cholestasis
Clinical Complications
Clinical manifestations of cholestasis are the consecuence of the accumulation of substances normally excreted in bile. Generally, they are visible later in the course than the biological manifestations. Jaundice is the most common clinical manifestation. Results from the bilirubin accumulation in the blood.
Pruritus is frequently observed, but hasn’t been well explained. May be secondary to the increase of bile acids in the blood, and/or other substances, particularly endorphins (JAMA 1992; 268: 3359-3362).
In cases of prolonged cholestasis, the accumulation of lipids (cholesterol > 11,6 mmol/L for over 3 months) leads to the formation of xanthomas, xanthelasma. Other manifestations are associated to the decrease of bile acids in the intestine which interrupts the enterohepatic cycle, leading to fat and liposoluble vitamins (A, D, E, K) malabsorption.
The evolution of chronic cholestatic diseases (primary biliary cirrhosis, primary sclerosing cholangitis) may be marked by the appearance of signs og hepatocellular insufficiency and portal hypertension, leading to liver transplantation.
Biological Complications
The earliest complication is the increase in the blood concentration of bile acids:
- Increase in bilirubin and cholesterol concentrations;
- Increase in the activity of alkaline phosphatase, 5′ nucleotidase, GGT;
- Prothrombine time prolongation initially secondary to the decrease of vitamin K dependent coagulation factors, then to a defcit of factor V which translates in hepatic insufficiency.
Histological Complications
Cholestasis is a microscopic accumulation of bilirubin inside the liver parenchyma. This stasis may be inside the hepatocyte, inside the kupffer cell or canalicular. It initiates in the centrolobular region.
Hepatocyte lesions in the peroportal region appear during prolonged cholestasis. Hepatocytary rossettes give the diagnosis of this type of lesion. Inside the parenchyma, hepatocytes are disposed around the lummen and contain an eosinophylic material, or bilairy pigment, giving a tubular appearance. On ocassions a neoductular proliferation, is responsable in part, for the increase in GGt activity.
The evolution of chronic cholestasis is marked by the development of fibrosis and then cirrhosis.
Molecular Mechanisms of Cholestasis
They are numerous. cloning of different transporters has allowed the better understanding of the molecular mechanisms of many hereditary diseases and at the same time, progress in the cholestatic mechanisms.
Familial Intrahepatic Cholestasis and Benign Recurrent Cholestasis
The familial intrahepatic cholestasis are a heterogenous group of cholestatic diseases occuring in children and lead in a short time to severe liver disease.
Three different entities have been identified (Types 1, 2 and 3). Benign recurrent cholestasis is a recessive autosomic disease, characterized by episodes of cholestatic jaundice, spontaneous pruritus without leading to chronic liver disease.
1. Cholestasis due to a modification in the symmetry of the canalicular membrane
Patients with Byler’s disease have been identified with having FIC1 gene mutations. The function of the codified protein is still hypothetical. It may be an aminophopholipid transporter, with a role in the maintenance of the asymmetry of the canalicular membrane.
The dissappearance of the asymmetry may compromise the function or insertion of transporters of this membrane, so they lead to cholestasis. It may as well be a transporter.
In patients with benign recurrent cholestasis, the mutated gene is also the FIC1 (Nat Genet 1998; 18: 219-224). But in these cases the mutation leads to partial inactivation of the protein while it’s fucntion would be severly compromised by the mutations observed in Byler’s disease.
2. Cholestasis due to a decrease of the canalicular secretion of bile acids
Familial intrahepatic cholestasis type 2, the SPGP protein is absent in the canalicular membrane. The bile acid secretion in these patients is inferior to 1%. Other mutations of FIC2 gene have been identified. Bile acids that accumulate in the hepatocyte are responsible of cellular lesions (Gastroenterology 1999; 117: 1370-1379).
3. Cholestasis secondary to a decrease in the phopholipid canalicular secretion
The familial intrahepatic cholestasis type 3 is related to mdr3 gene mutation. Phospholipid biliary secretion is suppressed while bile acid secretion is normal.
The micelles formed inside the biliary duct are simple micelles deprived of phospholipids and posess a strong detergent power. This power i exerciced by the cholangiocytes and hepatocytes. Due to membranous lesions, a ductular proliferation can be seen, explained in part by the increase in GGT activity (Hepatology 1998; 28: 530-536).
Arterio-Hepatic Dysplasia or Syndromic Interlobular Bile Duct Hypoplasia (Alagille’s Syndrome)
This is a genetic disease of dominant autosomic transmission, associated to a chronic neonatal cholestasis secondary to hypoplastic bile ducts, hypoplasia or stenosis of the pulmonary artery, defects in the posterior vertebral arch and a posterior embryotoxon.
Bile duct hypoplasia appear secondary to a mutation of Jagged1 gene which codifies a protein that plays a role in the transmission of intercellular information, after fixing itself on a NOTCH receptor in the cellular membrane (Hum Mol Gen 1998; 9: 1363-1369).
After this fixation, part of the receptor is translocated inside the nucleus where it joins an ADN binding protein, allowing the direct transmission of the signal produced by the nucleus. NOTCH controls other genes that intervene in cellular proliferation, differentiation and apoptosis.
Recently, it has been demonstrated that NOTCH receptor is absent in biliary cells during neoductular proliferation secondary to cholestasis. This receptor may then play a role in the formation of normal bile ducts (Hepatology 2001; 34: 1184-1192).
Cholestasis Secondary to Cytokine Secretion
A clinical cholestatic syndrome occurs frequently during infections, cancer, granulomatous diseases, macrophagic activation diseases. Histologically the liver architecture is preserved, but an intrahepatic cholestasis is present and an inflammatory infiltrate which may organize as granulomas.
There is a synthesis of certain cytokines such as IL-6, TNF-alpha, by Kupffer cells in response to sepsis and endotoxin liberation.
Obstructive Cholestasis
After CBD ligation in rats, bile acid concentration increases the first three days and then decreases progressively. This suggests an adaptation of different bile acid transporters during cholestasis. A decrease in transcription transporter has been demonstrated, while the expression of the canalicular transporter of bile acids is preserved.
Simetimes, the ileal trasnporter expression is decreased and the renal transporter increases (Gastroenterology 2001; 121: 1473-1484). These different adaptations allow the decrease of bile acid accumulation in the liver, as well as their toxicity.
Hepatobiliary Surgery and Liver Transplantation
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