**3. The biliary duct epithelium**

a collapsed PCL, concentration of mucins within the mucus layer, and adhesion of mucus

**•** *The high salt hypothesis* suggests that the ASL normally is hypotonic [84] and provides an optimal environment for defensins. According to this view the ASL in CF patients would have a higher salt concentration than normal because the absorbing function of ENaC depends on the state of CFTR and cannot be activated when CFTR is defective or absent [84].

**•** *The low pH hypothesis* focuses on the interactions between CFTR and the SLC26 and proposes an acidic ASL. This may compromise the function of airway immune cells and increase toxic oxidant species. Lowering the pH may also eliminate electrostatic repulsive charges between organisms and facilitate "tighter" biofilm formation as well as reduce electrorepul‐ sive forces between bacteria and negatively charged mucins. Furthermore, ciliary beat frequency in bronchial epithelium is reduced when external pH falls [85]. All the above factors may inhibit mucociliary clearance (MCC) and thus elimination of bacteria from the

**•** *The low oxygenation hypothesis* postulates that the oxygen content of the ASL is low, due to build-up of mucus plugs, resulting in enhanced growth of the facultative anaerobic

**•** *The defect gland function hypothesis* suggests that the primary defect in CF is reduced fluid secretion by airway submucosal glands and possibly altered secretion of mucous glycopro‐

**•** *The soluble mediator hypothesis* proposes that signalling molecules within the ASL itself are controlling ASL volume [89]. These molecules are ATP, which is breathing- or shear-stress induced [90], and adenosine. ATP interacts with receptors such as the purinergic P2Y2 receptors and adenosine reacts with the adenosine A2b receptors, that mediate inhibition of ENaC and activation of both CFTR and CaCC [91, 92]. This mechanism is also supposed

An interesting question is what the role of aquaporins (AQP) is in the production of ASL, compared to paracellular water flow and CFTR. In the epididymis, CFTR appears to regulate AQP-mediated water permeability [93]. In this tissue, CFTR is co-localized with AQP9 in the apical membrane, and this association promotes the activation of AQP9 by cAMP [94]. In a heavily debated study, concerning the clinical benefit of nebulized hypertonic saline in cystic fibrosis, an important role of amiloride-inhibitable AQP water channels in the generation of ASL was proposed [95]. However, although the positive effect of hypertonic saline as such is not disputed, the question whether this effect is mediated by AQP has received conflicting answers [96, 97] and is still open. Recently, it has been found that interleukin (IL)-13 enhances the expression of CFTR but abolishes the expression of AQP in airway epithelial cells [98]. In

The differences in the proposed hypotheses are due to difficulties in determining the accurate composition of the ASL because of the very small depth of the layer. Among the problems encountered there are difficulties to collect an adequate amount of ASL without disturbing the

to include PDZ interactions and cytoskeletal elements [1].

conclusion, the relation between CFTR and AQP needs further study.

to the airway surface [83].

8 Regenerative Medicine and Tissue Engineering

airways [86].

*P.aeruginosa* [87].

teins [88].

The biliary tree is a complex network of conduits within the liver that begins with the canals of Hering and progressively merges into a system of ducts, which finally deliver bile to the gallbladder and to the intestine. Cholangiocytes are the epithelial cells forming the biliary epithelium which shows a morphological heterogeneity that is strictly associated with a variety of functions performed at the different levels of the biliary tree [102]. Thus, the canals of Hering, located at the ductular-hepatocellular junction, constitute the physiologic link of the biliary tree with the hepatocyte canalicular system and they are the site where a facultative progenitor cell compartment resides; these liver progenitor cells are variably elicited only after liver injury. Given the strong capacity of mature hepatocytes to proliferate, cholangiocyte ability to behave as liver progenitor cells becomes evident only when hepatocellular proliferation is hampered as a result of severe liver damage, as that induced by several toxins or drugs, or occurring under certain conditions, *i.e.* viral hepatitis or non alcoholic steatohepatitis [103]. Cells lining the intrahepatic biliary tree have different functional and morphological specializations: the terminal cholangioles (size <15 μm) have some biological properties such as plasticity (i.e., the ability to undergo limited phenotypic changes) and reactivity (*i.e.*, the ability to participate in the inflammatory reaction to liver damage); interlobular (15-100 μm) and large ducts (100 μm to 800 μm) modulates fluidity and alkalinity of the primary hepatocellular bile.

#### **3.1. Ion and water transport in cholangiocytes**

In addition to funnelling bile into the intestine, cholangiocytes are actively involved in bile production. In humans, around 40% of the total bile production is of ductal origin. Cholan‐ giocytes exert a series of reabsorptive and secretory process which dilute and alkalinize the bile during its passage along the biliary tract. Modifications of ductal bile appear to be tightly regulated by the actions of nerves, biliary constituents, and some peptide hormones like secretin [104]. Accordingly to *in vivo* and *in vitro* models, it is possible to distinguish between three different bile flow fractions: 1) the canalicular bile salt-dependent flow that is driven by concentrative secretion of bile acids by the hepatocytes followed by a facilitated efflux of water; 2) the canalicular bile salt-independent flow, which is also created by hepatocytes but through active secretion of both inorganic (bicarbonate) and organic (glutathione) compounds; and 3) the ductal bile flow, that is the bile salt-independent flow contributed by cholangiocytes, mainly through production of a bicarbonate-rich fluid in response to secretin and other regulatory factors. Cl secretion into the ductal lumen is the driving force of a chloride/ bicarbonate exchanger that exports HCO3 into the bile flowing into the biliary tree. Indeed, this AE (anion exchanger) activity is facilitated by the outside to inside transmembrane gradient of Cl at relatively high intracellular concentrations of HCO3- , specially upon secretin stimulation. The AE activity in the liver is operated by AE2/SLC4A2 which is localized not only in the canaliculi but also in the luminal membrane of bile duct cells [105]. Experiments of RNA interference with recombinant adenovirus expressing short/small hairpin RNA have confirmed that AE2/SL4A2 is indeed the main effector of both basal and stimulated Na+ independent Cl- /HCO3 exchange in rat cholangiocytes [106]. Besides acid/base transporters cholangiocytes possess other ion carriers like those for Cl- , Na+ , and K+ , which greatly contrib‐ ute to intracellular pH regulation and bicarbonate secretion. Thus, CFTR had been localized at the apical side, where it plays a role in biliary excretion of bicarbonate [107, 108]. Although bicarbonate permeability through activated CFTR has been shown in several epithelia [109], its main contribution to biliary bicarbonate secretion appears to occur through a coordinated action with AE2/SL4A2 [106, 110, 111]. In addition to CFTR, cholangiocytes possess a dense population of Ca2+-activated Cl- channels. These channels are responsive to interaction of the purinergic-2 (P2) receptors with nucleotides (mainly ATP or UTP) [112, 113]. The apical fluxes of anions results in increased osmotic forces in the bile duct lumen which in the presence of AQPs contributes to water flux. AE2/SLC4A2 and CFTR colocalize with AQP1 in cholangiocyte intracellular vesicles wich coredistribute to the apical cholangiocyte membrane upon both cAMP and secretin stimulations [114].

#### **3.2. The pathogenesis of CF liver disease**

CF is associated with liver disease in almost 30% of all patients. In general, CF-associat‐ ed liver disease develops during the first decades of life and does not progress rapidly. The diagnostic criteria were initially established by Colombo et al. [115]. Hepatobiliary disease in CF encompass a wide variety of complications, including steatosis, focal biliary cirrhosis (FBC), multilobular biliary cirrhosis (MBC), microgallbladder, distended gallblad‐ der, cholelithiasis, intraheapatic sludge or stones, and cholangiocarcinoma [116]. The pathogenesis of steatosis (fatty liver) is not directly ascribed to the CFTR gene defect but has been attributed to malnutrition, essentially fatty acid deficiency, carnitine or choline deficiency, or insulin resistance [117].

With regarding to the pathogenesis of FBC and MBC, various hypotheses have been proposed [118, 119]:

**•** *The low chloride secretion hypothesis* proposes that loss of CFTR function leads to blocked biliary ductules with thick periodic acid-Schiff positive material leading to acute and chronic periductal inflammation, bile duct proliferation and increased fibrosis in scattered portal tracts. Hepatic stellate cells (important drivers of hepatic fibrosis) become activated to produce collagen and stimulate the bile duct epithelium to produce the profibrogenic cytokine TGF-β. The progression of FBC to MBC and portal hypertension, which occurs in up to 8% of patients, may take years to decades, and should be viewed as a continuum [120]. Considering CFTR as a driving force for Cl- /HCO3 exchange, the postulated sequence of CF-associated hepatobiliary complications is that loss of functional CFTR protein in the apical membrane of cholangiocytes presumably initiates a cascade of abnormal Cl- and HCO3 secretion, decreased bile flow, bile duct plugging by thickened secretions, and cholangiocyte/hepatocyte injury [10].

