Bile is about ninety-five percent water, yet tiny membrane channels help decide how that water moves through the liver, bile ducts, and gallbladder. This review asks whether those channels could become therapeutic targets.

Aquaporins are transmembrane proteins permeable to water and a series of small solutes. They play a key role in hepatobiliary secretion at the level of the liver, bile ducts, and gallbladder. AQP eight and AQP nine facilitate osmotic water movement in hepatic bile, while AQP one and AQP four help rearrange bile composition in the biliary tract.

In the gallbladder, AQP one and AQP eight support bidirectional water flow that ultimately concentrates bile, and their dysregulation has been associated with hepatobiliary disorders. Bile is mainly water and solutes, including cholesterol, phospholipids, and bile acids.

It is produced by the liver, concentrated in the gallbladder during fasting, and released into the duodenum after a fat-enriched meal and neurohormonal stimulus. In the intestine, bile supports digestion by increasing the emulsification and absorption of dietary lipids and fat-soluble vitamins.

It is also the main route for cholesterol excretion from the body. Figure one organizes the thirteen human aquaporins into overlapping functional groups: orthodox aquaporins, aquaglyceroporins, peroxiporins, and superaquaporins. It connects these proteins to physiological roles including fluid generation, metabolic balance, cellular functions, and immune responses, while also showing disease contexts such as metabolic syndrome, cardiovascular disease, cancer, and liver disorders.

The visual matters because it frames aquaporins as multifunctional regulators whose distribution and activity are relevant in both health and pathology. Aquaporins play a crucial role in maintaining metabolic homeostasis, and their distribution, expression, and function are regulated under normal and pathological conditions.

Aquaporin dysregulation has been implicated in metabolic syndrome, cardiovascular diseases, renal concentration disorders, obesity, diabetes, liver steatosis, and gallstones. In the hepatobiliary system, hepatocytes express AQP eight, AQP nine, and AQP eleven.

AQP eight has multiple subcellular localizations and roles including bile water secretion, ammonia detoxification, redox balance, and cholesterol biosynthesis. Cholangiocytes express AQP one, which participates in water flow during bile formation, and secretin regulates AQP one-facilitated water secretion through cyclic adenosine monophosphate signaling.

Gallbladder epithelial cells express AQP one and AQP eight, which contribute to water movement into and out of the gallbladder. Bile is about ninety-five percent water, and healthy adult humans secrete about zero point eight to one point zero liters of hepatic bile daily, at a rate of thirty to forty milliliters per hour.

Bile formation begins at the canalicular membrane of hepatocytes as an osmotic process. Bile acids and other constituents are actively secreted into bile canaliculi, creating the osmotic force for parallel water secretion. Bile flow also has a bile-acid-independent component, attributed to active secretion of osmotically active inorganic electrolytes and organic anions.

The epithelial cells lining the mammalian hepatobiliary tree express several aquaporins with distinct subcellular localizations and roles. Endothelial cells in the hepatobiliary system express AQP one. Table 1 maps AQP8 within the liver parenchyma, identifying hepatocytes in rat, mouse, and human, with localization at the APM, SAV, IMM, and SER.

It links these sites to canalicular bile secretion, cytoplasmic osmotic homeostasis, mitochondrial ammonia detoxification and ureagenesis, mitochondrial H2O2 release, cholesterol biosynthesis, and regulation of metabolic signaling. This matters because bile formation begins at the hepatocyte canalicular membrane as an osmotic process involving solutes and water.

Human and rodent hepatocytes express high levels of AQP eight and AQP nine, while mouse hepatocytes also express AQP eleven. Human hepatocytes additionally express AQP three and AQP seven. The redundancy of aquaporins in human hepatocytes remains unclear, although different molecular selectivity and subcellular localization may justify it.

The physiological meaning of AQP three and AQP seven in hepatocytes is unclear. In hepatocytes, AQP eight is found at the canalicular membrane, subapical vesicles, mitochondria, and smooth endoplasmic reticulum. It is reported to mediate canalicular bile water secretion and help preserve cytoplasm osmolarity.

AQP eight is also reported to facilitate ammonia movement in mitochondrial ammonium detoxification and ureagenesis, and hydrogen peroxide efflux from mitochondria during oxidative stress. Studies with Huh-seven cells and primary rat hepatocytes suggested a role for mitochondrial AQP eight in cholesterol biosynthesis modulated through the sterol regulatory element-binding protein.

Choleretic agonists such as dibutyryl cyclic adenosine monophosphate and glucagon trigger translocation of AQP eight-containing subapical vesicles to the canalicular plasma membrane through a phosphatidylinositol-three-kinase-dependent microtubule-associated pathway.

Insertion of AQP eight into the hepatocyte apical membrane increases canalicular plasma membrane water permeability and drives osmotic water movement into the bile canaliculus. Figure two proposes how aquaporins coordinate water movement during bile formation in hepatocytes.

AQP9 channels osmotic water from the sinusoidal bloodstream into the cell, while AQP8 directs water across the canalicular membrane into the bile canaliculus; smaller routes include lipid-bilayer diffusion and paracellular flow through tight junctions. The figure also links glucagon to cyclic AMP and PKA signaling, which promotes microtubule-dependent delivery of AQP8-containing vesicles to the canalicular membrane.

AQP nine is an aquaglyceroporin of broad selectivity that permits movement of water, glycerol, other polyols, hydrogen peroxide, urea, and several additional solutes. In rodent hepatocytes, AQP nine is the principal pathway for glycerol uptake from portal blood during fasting, and imported glycerol is converted into glycerol-three-phosphate, a major substrate for gluconeogenesis in early starvation.

AQP nine-depleted knockout mice have diminished liver glycerol permeability and enhanced plasma glycerol and triacylglycerol levels. Cholangiocytes produce secretin-induced ductal bile secretion through a cyclic adenosine monophosphate-dependent pathway. Activation of cystic fibrosis transmembrane conductance regulator drives bicarbonate extrusion through apical anion exchanger isoform two.

Bicarbonate and chloride provide the main driving force for osmotic water movement through apical AQP one into the biliary lumen. AQP one is present in subapical membrane vesicles with anion exchanger isoform two and cystic fibrosis transmembrane conductance regulator, and secretin regulates exocytic insertion of these vesicles into the apical membrane.

Figure 3 proposes how secretin stimulates ductal bile secretion in cholangiocytes. Through cAMP and microtubules, secretin promotes exocytosis of subapical vesicles containing AQP1, CFTR, and AE2 into the apical membrane.

Chloride exit through CFTR supports bicarbonate secretion through AE2, creating an osmotic force that moves water from the blood side, mainly through basolateral AQP4, into the bile duct lumen through AQP1. Water permeability did not decrease in cholangiocytes isolated from AQP one-depleted knockout mice.

Compensatory upregulation of other mouse cholangiocyte aquaporins, such as AQP eight, was hypothesized to explain this observation. Intrahepatic bile ducts both secrete and absorb water. Osmotic absorption is likely triggered by active absorption of sodium-coupled glucose and bile acids through the SGLT1 and ASBT cotransporters.

The gallbladder is a dynamic reservoir of diluted hepatic bile, and its concentration during fasting depends on water movement across the gallbladder epithelium. That movement is driven by osmotic gradients generated from active salt absorption and secretion.

Human and murine gallbladder epithelial cells express AQP one and AQP eight. AQP one is found at apical and basolateral plasma membranes in epithelial cells lining the gallbladder neck. AQP one is also found at the gallbladder corpus plasma membrane and over subapical vesicles that can be incorporated into the apical membrane through a microtubule-dependent, cyclic adenosine monophosphate-stimulated mechanism.

Figure four proposes how aquaporins move water across gallbladder epithelial cells during bile absorption and secretion. AQP8 is shown on the luminal side for osmotic water absorption, while AQP1 supports secretion into the gallbladder lumen and also appears in subapical vesicles that may reach the apical membrane through exocytosis.

Additional AQP1 channels on the basolateral side are proposed to facilitate water entry and exit, giving a mechanistic framework for gallbladder water transport. The exact physiological relevance of AQP one and AQP eight in gallbladder function remains debated because studies have reported discrepant results.

One study found similar bile acid concentrations in gallbladders from wild-type and AQP one-ablated mice, with no apparent functional substitution of AQP one by AQP eight. That observation was not consistent with earlier work linking diminished gallbladder concentrating function with decreased AQP one or AQP eight levels, so further targeted work is required.

Several diseases affecting the hepatobiliary tree are associated with abnormal bile fluid transport and cholestasis. Experimental models of cholestasis show deranged hepatobiliary aquaporins and bile secretion. Experimental models of extrahepatic obstructive, estrogen-induced, and sepsis-induced cholestasis suggest that dysregulated canalicular AQP eight contributes to cholestasis.

Downregulation of canalicular AQP eight is associated with decreased canalicular osmotic water permeability, and altered solute transporters and AQP eight function are likely to impair coupling between osmotic gradients and canalicular water flow. Adenoviral transfer of the human AQP one gene to rat liver improved bile flow in estrogen-induced cholestasis, suggesting potential therapeutic implications for cholestatic diseases.

Gallstone disease has a twenty percent prevalence in adulthood, and about eighty percent of gallstones are made of cholesterol. Its risk factors include insulin resistance, type two diabetes, visceral adiposity from overweight and obesity, and metabolic syndrome.

Gallbladder stasis, mucin hypersecretion, mucin gel accumulation, and immune-mediated gallbladder inflammation are additional promoting factors for cholesterol stone formation. During lithogenesis in mice susceptible to diet-induced cholesterol gallstones, reduced gallbladder AQP one and AQP eight expression was associated with reduced gallbladder concentrating ability.

AQP eight-depleted mice developed gallstones faster, and adenoviral liver expression of AQP eight or AQP one rescued that phenotype. In wild-type mice, scutellarin increased hepatocyte AQP eight expression, increased bile formation, reduced bile lipid concentrations, and prevented cholelithiasis compared with AQP eight knockout littermates.

Progress in developing drugs targeting aquaporins has been limited, partly because the AQP pore has been viewed as inherently resistant to drug targeting and because current experimental methods are difficult to reproduce. AQP-targeted drug development is nevertheless growing, with natural and synthetic compounds capable of selectively blocking channels or modulating aquaporin expression and regulatory mechanisms.

Preclinical cellular and animal studies show that synthetic and natural compounds can modulate aquaporins in different disease models. Potential strategies could target AQP one and AQP eight expression in the liver, cholangiocytes, and gallbladder epithelium. Modulating bile concentration could be useful in cholestasis and in people at high risk of gallstone formation.

Targeting AQP one and AQP eight could potentially improve bile flow, decrease symptoms and local inflammation, improve water homeostasis, and regulate bile volume. Figure five maps how aquaporins mediate water movement across hepatocytes, cholangiocytes in the bile ducts, and gallbladder epithelial cells, alongside bile flow toward the gallbladder lumen.

It highlights AQP1 and AQP8 as proposed targets for diseases that disrupt bile secretion or flow, with the aim of improving water homeostasis and regulating bile volume. The authors note that such modulation could be relevant to cholestatic liver disease, biliary inflammation, and gallstone risk.

Clinically relevant aquaporin-targeted therapies have been proposed, but clinical trials are still lacking and many difficulties remain. In the hepatobiliary system, aquaporins help maintain bile composition and flow, bile water secretion and reabsorption, and plasma glycerol uptake by hepatocytes and its conversion to glucose during starvation.

Basic and translational studies associate dysregulated aquaporin expression or function with hepatobiliary disorders including cholestatic liver diseases and cancer. Aquaporins help regulate bile water movement and are altered in disorders including cholestasis and gallstones.

AQP one and AQP eight are promising targets, but clinically tested therapies are still lacking.