Journal of Hepatology
Volume 39, Issue 5 , Pages 864-874, November 2003

Water handling and aquaporins in bile formation: recent advances and research trends

  • Piero Portincasa

      Affiliations

    • Section of Internal Medicine, Department of Internal Medicine and Public Medicine, Medical School, University of Bari, Bari, Italy
    • ,
  • Antonio Moschetta

      Affiliations

    • Section of Internal Medicine, Department of Internal Medicine and Public Medicine, Medical School, University of Bari, Bari, Italy
    • ,
  • Amelia Mazzone

      Affiliations

    • Department of General and Environmental Physiology, University of Bari, via Amendola 165/A. I-70126 Bari, Italy
    • ,
  • Giuseppe Palasciano

      Affiliations

    • Section of Internal Medicine, Department of Internal Medicine and Public Medicine, Medical School, University of Bari, Bari, Italy
    • ,
  • Maria Svelto

      Affiliations

    • Department of General and Environmental Physiology, University of Bari, via Amendola 165/A. I-70126 Bari, Italy
    • ,
  • Giuseppe Calamita

      Affiliations

    • Department of General and Environmental Physiology, University of Bari, via Amendola 165/A. I-70126 Bari, Italy
    • Corresponding Author InformationCorresponding author. Tel.: +39-0805-442928x2453; fax: +39-0805-443388

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1. Introduction 

Bile is formed by an osmotic process generated by ATP-dependent secretion of biliary constituents from the sinusoidal blood or the cellular interior into the bile canalicular lumen. Luminal accumulation of osmotically active solutes, in turn, induces the passive movement of water into the bile canaliculus [1], [2]. Canalicular bile (primary bile) is then modified by the bile duct epithelium via secretory and absorptive processes, stored and concentrated in the gallbladder (with few exceptions), and released into the gut.

In recent years, there has been considerable progress in understanding the molecular mechanisms underlying bile flow and many of the related solute transporters have been cloned and functionally characterized [3]. However, although the bulk of water generated at the lumen of the biliary tree is a major determinant of bile flow [4], relatively little attention has been given to the molecular mechanisms by which water is transported from the blood to the bile canaliculus or the bile duct. Moreover, studies aimed at answering the long-standing question on the route taken by the water transported across hepatocytes during primary bile formation have been limited and largely indirect. A paracellular pathway between adjacent cells was first evoked [5], [6]. Then, a transcellular route across the lipid bilayer or through discrete membrane proteins was favored [7], [8]. Knowledge of the phenomenon of water transport in bile formation and bile flow was boosted considerably following the recent recognition of multiple members of the aquaporin family of water channels (AQPs, aquaporins) variously expressed in liver epithelial barriers (for review see Ref. [1]). Recent works as described below indicate roles for aquaporins in liver physiology including bile elaboration and hepatic metabolism as well as in hepatic disorders with altered fluid homeostasis.

In this review, we summarize and evaluate the current concepts on the nature of hepatic water transport and present some information on what is known about aquaporins and their functional relevance in liver fluid balance and metabolism. Additionally, we discuss the emerging concepts about the significance of aquaporins in some clinical conditions of the hepatobiliary tract. Crucial areas for future basic and clinical research are also highlighted.

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2. Transport of water across the epithelial barrier: paracellular versus transcellular route 

Driven by an osmotic gradient, water may cross an epithelial barrier by two distinct routes: the paracellular pathway through tight junctions and the transcellular pathway through the cell plasma membrane and cytoplasm [9]. While it is generally accepted that the movement of water across tight epithelia (i.e. epithelia with high electrical resistance) occurs through a transcellular pathway [9] the route(s) adopted by water flowing through leaky epithelia (i.e. epithelia with low electrical resistance) is in dispute since the fraction of water that crosses the tight junction cannot yet be determined with confidence due to methodological limitations [9]. However, several studies have led to the conclusion that even in leaky epithelia the contribution of the paracellular pathway is negligible when compared to the transcellular pathway [10].

Regarding the transcellular route, the movement of water across plasma membrane may occur by simple diffusion through the lipid bilayer or movement through the aquaporin water channels [11]. A transmembrane transport of water by cotransporters (e.g. SGLT1, sodium-coupled glucose transporter) implying a secondary active water transport has been also proposed for some epithelia [12]. However, despite several papers supporting the water and solute cotransport hypothesis [12], unequivocal evidence has not yet been presented to invalidate the dogma that net water transport is proportional to the gradients of hydrostatic and osmotic pressure [13]. Membranes containing AQP proteins have proven to be 5–50-times more permeable to water than membranes lacking AQP water channels where the diffusional water movement strictly depends on the composition and fluidity of the membrane lipids [14]. The fact that lipid mobility is greater at increased temperatures explains why AQP-mediated water transport is relatively unaffected by temperature changes (Arrhenius activation energy <6 kcal/mol) compared to the diffusional movement through the membrane lipids (Arrhenius activation energy >10 kcal/mol). Diffusional movement of water occurs in all cells and is not pharmacologically inhibitable. How transcellular water moves through the cytoplasm is still undefined.

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3. The Aquaporin family of water channels: classification, structure, functional features, and known mechanisms of regulation 

Aquaporin channels are found in all living organisms, including archaea, eubacteria, fungi, protozoa, plants and all phyla of animals. They are small integral proteins of 25–34 kDa and belong to the broad superfamily of the Major Intrinsic Protein (MIP) transmembrane channels [15]. Nine of the 11 mammalian AQPs identified so far can be divided functionally into two major groups: AQP0–AQP2, AQP4 and AQP5, permeable to water but not to small neutral solutes (orthodox aquaporins), and AQP3, AQP7, AQP9 and AQP10, permeable to small uncharged solutes such as glycerol and urea in addition to water (aquaglyceroporins). AQP6 and AQP8, the other two mammalian AQPs, cannot be included in the above main groups due to functional and evolutionary constraints. AQP6 has an intracellular localization and exhibits extremely low water permeability which becomes of appreciable extent, curiously, after Hg2+ activation. Strikingly, when expressed in Xenopus oocytes, AQP6 features an anionic conductance (Cl− and nitrate) in response to acidic pH or Hg2+ treatment [16], [17]. On its part, AQP8 is marked by a divergent evolutionary pathway and unusual genomic organization when compared to the other mammalian AQPs [15], [18]. The reasons for such a distinction are still elusive and are awaiting investigation. Both rat and human AQP8 transport water [19], [20], [21], but mouse AQP8 was suggested to be permeable to urea in addition to water [22].

Structurally, AQPs are unique among channel proteins. Fig. 1a–d describes the biophysical structure of the most studied structural model of the AQPs, the orthodox AQP1. In plasma membrane, AQP1 is organized as homotetramer where each monomer contains an individual aqueous pore [23] (Fig. 1a). Topologically, each AQP1 monomer consists of six transmembrane α-helices connected by five connecting loops (A–E). Both the N- and C-termini are cytoplasmic (Fig. 1b). Connecting loops B and E partially deep the lipid bilayer and each contains the signature motif Asn-Pro-Ala (NPA) highly conserved among MIP channels. Loop A is N-glycosylated. Cys189 in loop E is the site of inhibition by the mercurial compounds, non-specific inhibitors blocking the activity of many transport proteins [24]. No specific pharmacological inhibitors of AQPs have as yet been described. Confirming the predicted ‘hourglass’ model [23] for aquaporin structure (Fig. 1c), the atomic structure of AQP1 was defined by cryoelectron microscopy to 3.8Å resolution [25] and then by X-ray analysis to 2.2Å resolution [26]. The architecture of the AQP1 channel consists of an extracellular and a cytoplasmic vestibule connected to a narrow pore (selectivity filter) (Fig. 1d). Within the selectivity filter the pore narrows to a diameter of 2.8Å, approximately the diameter of a water molecule which may permeate the pore in either direction. About half of the selectivity filter wall is quite hydrophobic whereas the other half is hydrophilic permitting a transient interaction (hydrogen bond) between four water molecules and defined pore-lining residues. This unusual combination establishes a selective pathway for coordinating an extraordinary amount of water transport (for the AQP1 individual channel, approximately 3×109 water molecules channel−1 s−1). The presence of a positively charged residue, Arg195, at the constriction region represents a barrier for protonated water permeation [26]. Glycerol permeation through aquaglyceroporins is similar to that of water. However, the wider permeability expressed by the aquaglyceroporin channel has been explained as being related to the larger diameter of the constriction region [26].

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  • Fig. 1. 

    Structural organization of the model orthodox aquaporin AQP1. (a) In plasma membrane, AQP1, the most studied structural model of AQPs, is a homotetramer, where each subunit contains an individual water pore. (b) Each aquaporin monomer consists of two tandem sequence repeats (repeats 1 and 2), each encoding three transmembrane α-helices with a short loop (hemipore; loops B and E) connecting the second and third helices. Both hemipore loops partially dip into the lipid bilayer and each contains the signature motif Asn-Pro-Ala (NPA) highly conserved among MIP channels. Both the N- and C-termini are cytoplasmic. (c) As predicted by the ‘hourglass’ model [23], the two repeats lie at 180° to each other and the aqueous pores results from the folding of the two hemipores into the membrane from opposite surfaces of the bilayer, overlapping midway through the bilayer where they are surrounded by six transmembrane helices. (d) The architecture of the channel within AQP1 was recently defined by X-ray crystallography to a 2.2 Å resolution [25], [26]. The channel consists of an extracellular and a cytoplasmic vestibule connected by an extended narrow pore (selectivity filter). Eight angstroms above the midpoint of the channel, within the selectivity filter, the pore narrows to a diameter of 2.8 Å (constriction site), a width sufficient to allow passage of water molecules in either direction [105], [106]. Residues at the constriction site, in particular histidine 182, are critical in establishing the water specificity of the AQP1 channel which even repels the protonated water (H3O+) [26]. Alternate residues are present at the constriction region of aquaglyceroporins, a feature responsible for the larger diameters and the consequent wider permeability exhibited by the related channels. Aquaporins are reversibly inhibited by Hg2+ which binds to cysteine residues flanking the aqueous pore (indicated in c). From King et al. [95], modified.

Gating does not seem to be a general feature of mammalian aquaporins, as they are generally constitutively active with no accessory proteins needed. Indeed, only one subset of AQPs is known to be gated. AQP0 is activated by low pH and inactivated by Ca2+ [27]. Moreover, while low pH inactivates AQP3, it activates AQP6 [17].

Regulation is physiologically critical for many channel proteins. Although the picture of the regulation mechanisms is certainly far from complete, several mammalian aquaporins are already known for being modulated on a short- or long-term basis. Long-term regulations of AQPs are often exerted at a level of gene transcription, as a result of specific signals brought by hormones, metabolites or β-adrenergic agonists. They generally modulate a specific AQP in certain tissues and at defined periods of fetal or postnatal life. AQP1 is upregulated by glucocorticoids in erythroleukemia cells, fetal proximal tubules and thin descending limbs of loops of Henle [28], [29], [30], [31]. Long-term stimulation of kidney AQP2 is exerted by vasopressin via V2 receptor followed by the dual effect of the cAMP signal on cAMP-responsive element binding protein (CREB) and immediate early gene expression [32]. Lithium [33], bilateral ureteral obstruction [34] and chronic hypokalemia [33] all cause marked reductions in renal AQP2 expression. By contrast, increases in AQP2 protein in the kidney collecting duct have been found in conditions of fluid retention including congestive heart failure [35], [36], cirrhosis [37] and pregnancy [38]. Mineralocorticoids increase AQP3 transcription in renal collecting duct [39]. AQP4 mRNA is induced by glucocorticoids and α-adrenergic agonists in perinatal rat lung [40]. AQP5 is induced in estrogen-treated, progesterone-primed uterus [31]. High plasma insulin suppresses the mRNA levels of an adipose-specific aquaglyceroporin, AQPap/7, and AQP9 in fat and liver, respectively, through the negative insulin response element (IRE) in AQPap/7 and AQP9 gene promoters [41]. AQP9 was reported to be upmodulated both by androgens and fasting in the adult rat epididymis [42] and liver [41], [43], respectively. On a short-term basis, several AQPs are regulated by exocytic insertion from an intracellular vesicular compartment into the plasma membrane in response to hormones or neurotransmitters via different second messenger and protein kinase/phosphatase. This allows for rapid adaptation (minutes) of fluid secretion/absorption, depending on physiological needs. In cholangiocytes, secretin induces the microtubule-dependent targeting of AQP1-containing vesicles to the apical plasma membrane via a cAMP second messenger [44]. This is a unique regulation for AQP1 among AQP1-expressing epithelia, as this AQP is usually expressed in a constitutive way. Under vasopressin challenge, AQP2 recycles between the intracellular compartment and the apical plasma membrane of chief cells of the renal collecting duct [45], [46]. AQP5 is translocated to the apical membrane of the acinar cells of rat parotid glands under acetylcholine action on M3 muscarinic receptors via cytosolic Ca2+ elevation [47]. As detailed below, in rat isolated hepatocytes, under active choleresis a pool of intracellular AQP8 redistributes to the canalicular membrane via a cAMP-mediated, microtubule-dependent mechanism [48], [49]. Insertion of functional AQP8 into the canalicular membrane of rat hepatocyte couplets was recently found to be induced by the choleretic hormone glucagon [50].

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4. Canalicular secretion of water 

Canalicular bile is mostly (>95%) made up of water [8], [51], [52]. Although large volumes of water are secreted into bile as a consequence of the osmotic driving force created by the active secretion of bile salts (BS) and other solutes (mainly glutathione) [53], [54], the molecular pathway underlying the movement of water from the sinusoidal blood to the bile canaliculus is a matter of debate. A paracellular route through tight junctions between adjacent hepatocytes was initially evoked, although direct experimental data were limited and largely indirect [5]. It was then indicated that canalicular secretion of water occurred across a transcellular route, mainly by diffusion through the plasma membrane lipids via a non-channel mediated pathway [7]. This hypothesis was based both on the fact that (i) the measured osmotic permeability coefficient (Pf) of isolated hepatocytes was even higher than that of cholangiocytes (66.4 cm−4 s−1 vs. 50.0 cm−4 s−1, respectively), which had been already shown to transport water via a transcellular pathway, and (ii) the calculated Arrhenius activation energy (Ea) associated to the hepatocyte water transport was much higher (Ea>12.8 kcal mol−1) than that characterizing the channel-mediated water transport (Ea<6.0 kcal mol−1). Recently, although confirming the transcellular pathway we and others found this hypothesis to be inconsistent with the presence of the mRNA of at least three distinct aquaporin water channels in rat, mouse and human hepatocytes: AQP0 [49], AQP8 [19], [20], [49] and AQP9 [49], [55], [56]. Also, a transcellular route via an AQP pathway is also indicated by the fact that the agonist-induced canalicular secretion in isolated hepatocytes is blocked by HgCl2 and dimethylsulfoxide (DMSO) [49], two compounds known to inhibiting AQP-mediated water transport. Thus, AQPs may play a central role in agonist-induced canalicular bile secretion by hepatocytes. The aquaporins expressed in the hepatobiliary organs and related selected biophysical, regulatory and genomic characteristics are listed in Table 1, Table 2, respectively.

Table 1. Reported cellular distribution of mammalian AQPs in the hepatobiliary system
SpeciesAQP expressedCellular locationSubcellular locationReferences
ManAQP1EndotheliumAPM, BLPM[100]
RatAQP0HepatocyteIC[49]
AQP1EndotheliumBLPM[85], [100]
CholangiocyteAPM, BLPM
AQP4CholangiocyteBLPM[101]
AQP8HepatocyteIC, APM[48], [49], [57], [59], [61]
AQP9HepatocyteBLPM[65]
MouseAQP1CholangiocyteNot determined[81]
AQP8HepatocyteIC, APM (?)[22]a

APM, apical plasma membrane; BLPM, basolateral plasma membrane; IC, intracellular location.

a Calamita et al., unpublished observations.

Table 2. Selected biophysical, biochemical and genomic characteristics of hepatobiliary AQPs
AquaporinTransported moleculesHgCl2 inhibitionGlycosylation consensusPKA/C consensusChromosomal localizationReferences
AQP0WaterYesNono12q13 (h)[60], [73]
AQP1WaterYesYesPKA (cryptic?)7p14 (h)[102]
AQP4WaterNoNoPKA, PKC18q11.2-12.1 (h)[78], [103]
AQP8Water, urea (m)YesYesno16p12 (h), 7F13 (m)[18], [19], [20], [22]
AQP9Water, glycerol, variety of non-charged solutesa, arseniteYesYesPKC15q22.1 (h)[55], [64], [104]

PKA, protein kinase A; PKC, protein kinase C; h, human; m, mouse.

a Urea, thiourea, polyols (glycerol, mannitol, sorbitol), purines (adenine), pyrimidines (uracil, 5-fluorouracil).

In rat hepatocytes, AQP0, AQP8 and AQP9 have a distinct subcellular expression, being differentially sorted in the hepatocyte compartments (Fig. 2) [57], [58], [59]. AQP0 (previously referred to as MIP) known for being the major intrinsic protein of lens fiber cells and featuring a relatively low permeability to water [60], was localized in intracellular vesicles mainly of rat pericentral hepatocytes [49]. AQP8 was found by us and, independently, other groups to be largely localized in membrane systems within the cell interior of hepatocytes [48], [57], [59], [61]. However, by immunoelectron microscopy, we also found some AQP8 presence in the rat canalicular plasma membrane [57]. A comprehensive immunohistochemical pattern of subcellular AQP8 distribution in rat hepatocytes is shown in Fig. 2a. Interestingly, Garcia et al. [48] and Huebert et al. [49], based on studies with rat cultured hepatocytes and hepatocyte couplets, respectively, reported that upon exposure of a choleretic stimulus, AQP8 redistributes from an intracellular vesicular compartment into the canalicular membrane via a microtubule- and cAMP-dependent mechanism. Based on their previous studies [7], and the observation that the above AQP inhibitors are ineffective on basal (absence of choleretic stimulus) canalicular secretion, Huebert et al. concluded that hepatocyte water movement is non-channel mediated in the basal state whereas it occurs through AQP during primary bile secretion [49]. Recently, this hypothesis was corroborated by studies with rat hepatocyte couplets suggesting that glucagon, an hormone known for having a cholecretic effect [62], [63], induces the protein kinase A and microtubule-dependent targeting of vesicles containing AQP8 to the canalicular plasma membrane by leading to a significant increase in membrane water permeability [50]. However, although appealing, this working model has to be further verified in the intact perfused liver. By immunohistochemistry, we recently observed that AQP8 maintains its predominant intracellular localization in hepatocytes of intact perfused liver even under active choleresis (Calamita G, Mazzone A, Svelto M, unpublished observations). Hence, it is conceivable that AQP8 might play additional roles, such as being involved in the osmoregulation of the hepatocyte cytoplasm and its vesicle/organelle content, being part of the signaling system that determines the hepatocyte hydration state (hepatocyte water content) or mediating the movement of water (or small neutral solutes) across intracellular membrane systems involved in hepatocyte metabolism. The possibility of multiple roles for AQP8 in hepatocytes is supported by our recent evidence of at least two alternatively spliced isoforms of AQP8 in rat and mouse liver (Calamita G, Mazzone A, Cassano G, Svelto M, unpublished observations). AQP9, an aquaglyceroporin characterized by also being permeable to a wide variety of neutral solutes including glycerol, carbamides, polyols, purines, pyrimidines and arsenite [55], [64], was localized exclusively in the hepatocyte basolateral membrane [58] where it was unaffected by the choleretic stimulus [49], [65]. A representative pattern of immunofluorescent localization of AQP9 in rat liver is reported in Fig. 2b. AQP9 expression in liver was suggested to be sex-linked, as male rats showed higher levels of both AQP9 mRNA and protein compared to the liver of female rats [66]. AQP9 may have a role in mediating the osmotic movement of water between the sinusoidal blood and the hepatocyte cell interior (Fig. 3). This function may relate to the formation of primary bile and/or provide the molecular pathway for the rapid shifts of water across, into, or out of the hepatocyte underlying the hepatocellular hydration state, an important and efficient mechanism of short-term control of canalicular secretion [67]. Indeed, a strict correlation has been demonstrated between the transcellular transport of BS across hepatocyte and the hepatocyte hydration state, as canalicular excretion of the bile salt taurocholate is stimulated by hepatocyte cell swelling and, conversely, inhibited by hepatocyte shrinkage [68]. Liver cell hydration can change within minutes under the influence of hormones, oxidative stress, and substrate supply to the liver [67]. Short-term modulation of hepatocyte cell volume is also a potent signal influencing cellular metabolism and gene expression [69]. Cell hydration may also relate to a hepatoprotective effect against a variety of noxes [70], [71]. As suggested by Tsukaguchi et al. [55], AQP9 may also provide the exit route for the urea produced within the hepatocyte or solutes, such as purines and pyrimidines derived from nucleotide synthesis de novo, lactate and ketone bodies. However, other urea transporters have been already described in liver [72]. Because fasting and re-feeding of mice increases and decreases hepatic AQP9 mRNA, respectively, and insulin deficiency results in an increase of the AQP9 transcript levels, Kuriyama et al. [41] and Carbrey et al. [43], hypothesized that AQP9 is the hepatocyte entry pathway for the plasma glycerol employed as a major substrate for hepatic gluconeogenesis. Moreover, based on the observation that in the rat pathological state of insulin resistance, the increased liver AQP9 mRNA, despite hyperinsulinemia, augments the utilization of glycerol for hepatic glucose production, Kuriyama et al. also suggested that the aquaglyceroporin AQP9 is of crucial importance in determining the glucose metabolism in physiology and insulin resistance [41]. Whether the major role of hepatic AQP9 is that of facilitating hepatocyte glycerol influx and urea efflux with a minor function in taking up water from sinusoidal blood will likely be deduced by the phenotype expressed the transgenic AQP9 knockout mice being presently generated. The physiological meaning of AQP0 in hepatocytes is presently obscure. Huebert et al. speculated that the role of AQP0 in hepatocytes may be unrelated to canalicular bile secretion [49]. In lens fiber cells, AQP0 has been suggested to have a structural function by facilitating cell-to-cell adherence between adjacent cells [73].

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  • Fig. 2. 

    Immunocytochemical localization of AQP8 and AQP9 in fed rat liver. (a) By confirming previous studies by us [57] and other groups [59], [61], immunohistochemistry using affinity purified rabbit antibodies directed against a specific rat AQP8 peptide (carboxy-terminus), strong AQP8 labeling is predominantly observed within the cell interior of most hepatocytes. AQP8 immunoreactivity is observed over the canalicular membrane of some hepatocytes (inset, arrow). (b) Immunofluorescent localization of AQP9 in rat liver by using affinity purified rabbit antibodies developed against a carboxy-terminus peptide of rat AQP9. In line with previous works [65], [66], clear AQP9 immunostaining is seen associated with the hepatocyte sinusoidal membrane (inset). No labeling is observed over the canalicular membrane (inset, arrows). Overall, labeling seems stronger in the peri-centrolobular region of the hepatic lobule than in the peri-portal area. Bars: a and b, 30 μm; insets in a and b, 12 μm. CV, centrolobular vein. The immunohistochemical experiments were performed with the kind contribution of Profs. D. Ferri and G.E. Liquori.

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  • Fig. 3. 

    The link between bile salt uptake and secretion and water transport in rat hepatocyte. Bile secretion by hepatocytes involves the active transport of solutes (primarily bile salts) followed by the passive movement of water into the lumen of the bile canaliculus. Sinusoidal water would be taken up by AQP9, an aquaglyceroporin selectively expressed in the hepatocyte basolateral plasma membrane, while the subsequent movement from the hepatocyte cell interior into the canaliculus lumen would be mediated by AQP8. AQP8 would be translocated from an intracellular pool (a) to the canalicular membrane under cAMP-mediated active choleresis [48], [49]. The choleretic hormone glucagon has recently been showed to induce insertion of AQP8 into the canalicular membrane of rat hepatocyte couplets [50]. However, AQP8 might play additional functions within the hepatocyte as suggested by the existence of a pool of AQP8 (b) constitutively expressed in an intracellular membrane system (Calamita G, Mazzone A, Svelto M). In addition to the entry of sinusoidal water, AQP9 has also been suggested to mediate the uptake of blood glycerol, a major substrate of hepatic gluconeogenesis, and represent the exit route for the urea produced within the hepatocyte. The function of AQP0, an aquaporin found in the intracellular compartment of hepatocyte, is obscure. See text for more details. BSUS summarizes the bile salt uptake systems (e.g. Ntcp); BSES summarizes the bile salt export systems (e.g. BSEP). The newer nomenclature for Ntcp and BSEP is SLC10A1 and ABCB11, respectively [107].

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5. Ductal bile secretion and absorption 

Bile is produced by two independent liver cell types, hepatocytes and cholangiocytes, the intrahepatic bile duct epithelial cells [52]. Although cholangiocytes represent only 3–5% of the liver cell population, they have a considerable role in bile formation, being capable of carrying out a variety of both secretory and absorptive functions. Indeed, cholangiocytes can contribute up to 40% of the daily output of bile fluid, depending on the species [74]. Ductal bile is a fluid particularly rich in HCO3− and Cl− which is stimulated by the gastrointestinal hormone secretin and, possibly, other agonists such as bombesin and vasoactive intestinal peptide (VIP) via specific receptors on the basolateral domain of cholangiocytes [51]. Hormonal stimulation increases the intracellular levels of cAMP which triggers a complex process, leading to the excretion of HCO3− and Cl−, followed by the osmotic movement of water into the ductal lumen [75]. Although knowledge of the molecular mechanism involved in bile duct secretion has been progressing rapidly in recent years [76], limited information is available on the stimulus-secretion coupling mechanism. After reporting the secretin-induced increase of AQP1 in the apical plasma membrane of cholangiocytes [44], Marinelli and LaRusso proposed a model to explain the bi-directional coupling of solute and water transport in rat cholangiocytes [8]. In this working hypothesis, secretin stimulates (via cyclic AMP) the exocytotic insertion of vesicles containing the cystic fibrosis transmembrane conductance regulator Cl− channel (CFTR), Cl−/HCO3− exchanger (AE2 isoform) and AQP1 water channel into the apical membrane of cholangiocytes. The efflux of Cl− via CFTR, in turn, provides the luminal substrate to drive the extrusion of HCO3− into the ductal lumen via AE2. Accumulation of HCO3− and Cl− in the biliary lumen creates the driving force for the osmotic movement of water via AQPs into the ductal lumen. Supporting the idea of a coupled water and electrolyte transport in rat ductal bile secretion, Tietz et al. recently provided evidence that rat hepatocytes contain a subpopulation of novel vesicles that sequester AQP1, CFTR and AE2 in basal state; these vesicles would move to the apical plasma membrane in response to secretory agonists [77]. Although AQP1 in rat cholangiocytes is present in both the apical and basolateral plasma membrane [75], AQP1 has been proposed to account only for the apical transport of water whereas AQP4, an aquaporin highly permeable to water [78] and restricted to the basolateral membrane of cholangiocytes, has been suggested as the main channel in mediating the uptake of water from the peribiliary vascular plexus surrounding the bile duct to the cell interior of rat cholangiocytes [1], [79]. A role for AQP1 in osmotically-driven apical water secretion during secretin-regulated ductal bile formation was also proposed by recent inhibitory studies in isolated rat and mouse bile duct units [79], [80]. Nevertheless, AQP1 null mice were reported to express a dietary fat processing defect whereas bile flow rates and bile salt concentrations were comparable to those of matched wild-type mice [81]. Such a controversy also resulted from recent works using protein knockout technology. Thus, while rat perfused bile duct units in which aquaporin-1 was knockout using small interfering RNA, supported the idea of a key role of aquaporin-1 in rat ductal bile formation [82], studies in bile duct units isolated from AQP1 knockout mice do not support a major role for water channels in bile secretion [83]. Because the mRNAs of multiple AQPs in rat cholangiocytes have been described in a recent preliminary report [84], further studies are needed to fully clarify whether AQP1 (or other AQPs) is rate-limiting for transcellular water movement in the bile duct epithelium and whether other AQPs are upregulated in cholangiocytes of AQP1 null mice. Paracellular movement of Na+ and K+ (and, consequently of water) into the lumen could also be generated by the negative intraluminal potential created by Cl− secretion. However, the extent to which water follows Na+ and K+ via a paracellular route is uncertain [1]. Roberts et al. [85] concluded that the specific contribution of the paracellular pathway to ductal secretion of water is not important, because the osmotic movement of water into rat bile duct lumen could not be inhibited by protamine, an agent known to block the paracellular pathway in certain tissues. The cholestatic hormone somatostatin has been reported to inhibit secretin-induced hypercholeresis and exocytosis by cholangiocytes [86], an observation in line with the regulatory effect exerted by this hormone on ductal bile secretion [87]. A working model for the solute and water transports in rat cholangiocytes is shown in Fig. 4.

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  • Fig. 4. 

    A model for the coupling of solute and water transport and somatostatin-induced absorption of ductal bile in rat cholangiocytes. The increase in intracellular cAMP induced by the choleretic hormone secretin would activate CFTR and lead to the exocytotic insertion of AQP1 in the cholangiocyte apical membrane. The efflux of Cl− would lead to the extrusion of HCO3− (via the Cl−/HCO3− exchanger, AE2) and Na+ and possibly K+ (through a paracellular pathway). The osmotic gradient created by these solute transports would drive a transcellular movement of water into bile duct. The existence of a sub-population of vesicles (asterisk) containing transporters involved in bile duct secretion including CFTR, AE2 and AQP1 has been recently suggested in rat cholangiocytes [77]. This vesicles would translocate exocytotically into the apical membrane under secretin challenge. Water would be taken up mostly by AQP4 and secreted into the lumen by AQP1. As stated in the text, the role played by AQP1 in mouse ductal bile secretion is currently controversial [82], [83], and further studies are awaited. Somatostatin would decrease the ductal bile secretion by reducing the intracellular levels of cAMP and inhibiting the exocytotic insertion of AQP1 and the activation of CFTR in the apical membrane. The net water ductal absorption caused by somatostatin would be the consequence of stimulating glucose and bile salt absorption by the Na-coupled glucose transporter (SGLT1) and apical sodium-coupled bile salt transporter (ASBT), respectively. The sodium/hydrogen exchanger isoform NHE3 has also been suggested to be involved in bile duct absorption [94]. See text for more details.

It has been suggested that the ATP released into bile by hepatocytes exerts a number of effects on bile duct epithelia, including that of stimulating cholangiocytes to secrete fluid and electrolytes via changes in cytosolic Ca2+ [88]. These actions are thought to result from stimulation of P2Y receptors expressed on the apical membrane of cholangiocytes. The ATP-induced secretion of bicarbonate in bile-duct epithelia has been suggested to result from serial activation of Ca2+-dependent chloride channels, then Cl−/HCO3− exchange [89]. Water would passively follow the bicarbonate secreted into the bile duct lumen. Interestingly, the bile salt ursodeoxycholic acid (UDCA) has been found to stimulate the release of ATP into bile by hepatocytes [88]. This led to the attractive hypothesis that the stimulation of bile-flow by ATP underlies the therapeutic action of UDCA on the hepatic manifestations of cystic fibrosis and other secretory disorders of the liver [88].

Recent studies with microperfused isolated rat and mouse intrahepatic bile ducts showed that intrahepatic bile ducts not only secrete but also absorb water [90]. Most of the driving force underlying the ductal absorption of water would be generated by the uptake of conjugated BS (cholehepatic circulation of BS) and glucose by the Na+-dependent bile acid transporter ASBT and Na+-coupled glucose transporter SGLT1, respectively, expressed by cholangiocytes [91], [92]. The absorption of biliary glucose would be related to the regulation of ductal bile formation [92]. SGLT1 could also carry water along with their substrates [12]. However, as seen above, unequivocal experiments supporting such a feature for SGLT1 are lacking. Reabsorption of glucose and/or BS by cholangiocytes has been suggested to be stimulated by the cholestatic hormone somatostatin [86], [93]. A recent work provided evidence for the presence of the sodium/hydrogen exchanger isoform NHE3 in the mouse and rat cholangiocyte apical membrane where this transporter was demonstrated to be functionally involved in the agonist-induced absorption of ductal bile [94]. Investigation is now required to assess the role of apical NHE3 in the regulation of bile secretion. A working model of fluid absorption in rat cholangiocytes is depicted in Fig. 4.

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6. Pathophysiology of aquaporins: relevance in bile flow disease 

Involvement of AQPs in human disease states is a rapidly emerging new area of investigation [95]. A variety of clinical conditions affecting the hepatobiliary system have been associated with abnormal fluid transport and consequent cholestasis [4]. Although understanding of the physiology of aquaporins in the hepatobiliary tract is at present only rudimentary, hepatobiliary AQPs have already been found to be dysregulated in clinical conditions including forms of extrahepatic cholestasis, insulin resistance syndrome and liver cirrhosis. It becomes apparent that AQP biology will prove relevant to the pathophysiology and perhaps even therapy of a large array of hepatobiliary diseases.

Our recent collaborative work using bile duct ligated (BDL) rats provided evidence that BDL-induced extrahepatic cholestasis causes post-transcriptional downregulation of AQP8 hepatocyte protein expression [96]. Besides corroborating the hypothesis of a role of AQP8 in canalicular water secretion, this indicated that defective expression of AQP8 water channels contributes to bile secretory dysfunction of cholestatic hepatocytes.

Dysregulated increases in liver AQP9 and fat-specific AQPap/7 have been suggested to underlie the pathophysiology of hyperglycemia in severe insulin resistance [41]. As argued by the Authors, the observed coordinated augmentation of the AQPap/7 and AQP9 transcription in insulin resistance increases the supply of fat-derived glycerol as a substrate for hepatic gluconeogenesis, which aggravates hyperglycemia. Future studies to identify the transcriptional factors and associated proteins involved in the transcriptional regulation of AQPap/7 and AQP9 genes by insulin may provide valuable insights to help design novel pharmacological strategies to treat insulin resistance syndrome.

Increased levels of plasma vasopressin and the consequent impaired ability to excrete water with urine are an important pathophysiological mechanism underlying the water retention associated with hepatic cirrhosis [97]. Consistent with this, the mRNA and protein levels of AQP2, the vasopressin-induced water channels inserted into the apical membrane of kidney collecting-duct principal cells under hormonal stimulation, were reported to increase in rats developing carbon tetrachloride-induced liver cirrhosis [37]. Administration of antagonists of V2, the vasopressin receptor, improve solute-free excretion in rats with cirrhosis and ascites [98]. However, the expression of AQP2 was found to vary considerably depending on the models of hepatic cirrhosis, because AQP2 (in addition to AQP3 and AQP4, the two AQPs located in the basolateral plasma membrane of kidney collecting-duct principal cells) was found to be down-regulated in rats with cirrhosis induced by common duct bile ligation [99]. Nielsen et al. argued that down-regulation of AQP2 occurs in compensated cirrhosis (peripheral vasodilation and increased cardiac output) by representing a physiological down-regulation to prevent the development of water retention, whereas increased vasopressin levels in cirrhosis with severe ascites are responsible for the up-regulation of AQP2 and hence increased water reabsorption.

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7. Conclusions and future perspectives 

Major advances have been achieved during recent years in all aspects of hepatobiliary physiology and pathophysiology, and the acquisition of new information resulting from the flurry of investigative activity is daunting to both scientists and clinicians alike. The recent recognition of aquaporins in all hepatobiliary epithelia is highly instructive, providing exceedingly valuable insights into the molecular pathways, biophysics and regulation of water movement in hepatobiliary epithelia. The existence of a coordinated network for transepithelial water movement in the hepatobiliary tract is anticipated. While a role for AQPs in facilitating canalicular and ductal bile secretion is already apparent, the current understanding of the regulation, physiology and pathophysiology of hepatobiliary AQPs is still in its infancy. Investigation should be addressed to studying the possible participation of AQPs in the sinusoidal uptake of water by hepatocytes, hepatocellular hydration state, exit of catabolic urea or other small neutral catabolic solutes out of the hepatocyte, uptake of blood glycerol, secretin-stimulated ductal bile secretion and somatostatin-induced net ductal absorption of water. The regulatory role of nuclear receptor FXR, the intracellular bile acid sensor, on the transcription of hepatocyte AQPs needs to be evaluated. The meaning of the AQP8 fraction constitutively expressed within the hepatocyte cell interior as well as the potential involvement of AQP8 and AQP9 in the liver glucose metabolism deserve attention. Specific association of AQPs with hepatobiliary disorders with altered fluid balance such as cholestasis, liver cirrhosis, cystic fibrosis, diabetes and polycystic kidney disease awaits further analysis. Valuable information will likely be provided by the aquaporin null phenotypes expressed by the multiple AQP knockout mice currently under characterization. Overall, this process should greatly enhance our understanding of bile physiology and pathophysiology and lead to novel therapeutic approaches in liver diseases featuring altered fluid homeostasis.

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PII: S0168-8278(03)00294-0

doi:10.1016/S0168-8278(03)00294-0

Journal of Hepatology
Volume 39, Issue 5 , Pages 864-874, November 2003

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