Aquaporin gene therapy for disorders of cholestasis?
Authors: Lukmanee Tradtrantip, Alan S. Verkman
Journal: Hepatology (2016)

Potential conflict of interest: Nothing to report. See Article on Page 535 The involvement of aquaporin (AQP) water channels in epithelial fluid transport is supported by compelling evidence, largely from transgenic AQP knockout mice and some from humans with loss‐of‐function mutations.1 It is instructive to think about the role of AQPs in epithelial fluid transport in three scenarios, as depicted in Fig. 1.Figure 1: Roles of aquaporin (AQP) water channels in epithelial fluid transport. (A) Glandular secretion. AQPs in acini of epithelial cells, as in salivary gland, increase water secretion in response to osmotic gradients produced by salt transport. (B) Transepithelial water transport. AQPs in epithelial cells lining kidney collecting duct increase water transport out of the lumen in response to an extrinsically generated osmotic gradient. (C) Near‐isosmolar fluid transport. AQPs in epithelial cells lining kidney proximal tubule facilitate water absorption in response to osmotic gradients produced by local solute absorption. Abbreviation: mOsm, milliosmoles.In glandular fluid secretion (Fig. 1A), as accomplished by salivary and airway submucosal glands, AQPs facilitate water secretion driven by osmotic gradients produced by active solute transport. Fluid secreted into the glandular acinus is then propelled into an adjoining duct by hydrostatic forces. Reduced acinar water permeability impairs glandular fluid secretion because of reduced osmotic water secretion into the acinar lumen. For example, saliva secretion is greatly reduced and its osmolality is increased in mice lacking AQP5,2 a water channel normally expressed at the apical (luminal) membrane of acinar epithelial cells in salivary gland. A second scenario (Fig. 1B) is osmotically driven water absorption across an epithelium in which the osmotic imbalance is created by extrinsic mechanisms, as occurs in kidney collecting duct. Luminal (urinary) fluid moves though the collecting duct and is exposed to osmotic gradients because of the high interstitial osmolality of the renal medulla. AQPs facilitate water absorption from the tubule lumen into the renal medulla, allowing the formation of a concentrated urine. Humans with loss‐of‐function mutations in apical membrane AQP2 show greatly impaired urinary concentrating ability (nephrogenic diabetes insipidus).3 A third scenario (Fig. 1C), which is most relevant to bile secretion, is “near‐isosmolar” fluid transport across an epithelium in which small osmotic gradients produced by solute pumping drive water movement locally, resulting in net transepithelial fluid transport. An example is the kidney proximal tubule in which active solute (NaCl, glucose, etc.) absorption creates an osmotic gradient that drives water absorption. Fluid absorption can be modulated by epithelial water permeability if fluid transport through the tubule is sufficiently rapid such that solute absorption creates an osmotic imbalance along the tubule. Osmotic water permeability in kidney proximal tubule is reduced ∼5‐fold by AQP1 deletion in mice, and fluid absorption is reduced by ∼2‐fold, which impairs urinary concentrating function.4 The idea of AQP gene delivery to increase epithelial water permeability and organ function was first reported in kidney, in which intravenous delivery of an adenovirus encoding AQP1 produced patchy AQP1 expression in proximal tubules of AQP1 knockout mice and a small increase in urinary osmolality.5 AQP gene delivery to salivary gland by retroductal infusion of an adenovirus encoding AQP1 improves saliva secretion in experimental animal models, and a phase I clinical trial has been completed for radiation‐induced salivary hypofunction.6 The articles by Marrone et al., in this issue of hepatology7 and an earlier paper,8 report that delivery of an adenovirus encoding AQP1 by retrograde infusion into the bile duct increases bile flow in a rat model of cholestasis produced by estrogen administration. The rationale for this approach is the reduced expression of AQP8 in cholestasis, a water channel expressed in hepatocyte canalicular plasma membranes. The logic is that increasing water permeability would increase osmotically driven water secretion from hepatocytes into bile canaliculi. Marrone et al.8 reported an ∼50% increase in bile flow in the adenovirus‐treated rats and a similar increase in “choleretic efficiency” related to the coupling between bile flow and bile salt secretion. Though the rationale may appear plausible that increased bile flow would follow increased canalicular water permeability, the result is actually quite unexpected from basic principles of fluid transport physiology. The issue is that the small dimension of bile canaliculi of ∼1 μm, which translates to a very high surface‐to‐volume ratio (>50‐fold greater than in kidney tubules), mandates rapid local equilibration of osmotic gradients produced by salt secretion. Rapid osmotic equilibration would occur even in the absence of AQPs, given that membranes have considerable AQP‐independent water permeability attributed to paracellular water flow and water transport through membrane lipids and non‐AQP proteins. A further increase in water permeability by AQP gene transfer would therefore not increase bile flow. Marrone et al.8 found that after adenovirus treatment, only ∼20% of hepatocytes expressed AQP1, and did so in a largely nonpolarized plasma membrane pattern, which raises further uncertainty in how AQP1 gene delivery could increase bile flow. If AQPs are not necessary for canalicular fluid secretion, then an obvious question is why do hepatocytes express them. Though the answer is not known, there are many examples, such as in lung and gallbladder epithelia,9 where transepithelial osmotic water permeability is AQP dependent, but near‐isosmolar fluid secretion is AQP independent, which is explicable on the basis of relative rates of solute transport and osmotic equilibration. Perhaps AQP function in certain tissues was needed in ancestral animals, or simply that AQP biology remains incompletely understood. Of relevance here, AQP8 deletion in mice did not produce demonstrable phenotypic abnormalities.10 A second major unexpected finding reported by Marrone et al. is the increase in bile salt secretion with AQP1 gene transfer. Little change in electrochemical driving forces for bile salt secretion is predicted with AQP gene transfer. Marrone et al.7 report that expression of the canalicular bile salt export pump (BSEP; ABCB11) is not altered by AQP1 gene transfer in rats with estrogen‐induced cholestasis, but its activity is increased by ∼2‐fold. Biochemical evidence suggested that the membrane microdistribution of BSEP is altered with AQP1 gene transfer from low cholesterol nonraft to high‐cholesterol raft microdomains, which might increase BSEP function. Though this proposed mechanism is novel and interesting, how AQP1 expression could produce a microdomain redistribution was not investigated, nor was it explained how AQP1 expression in only ∼20% of hepatocytes could fully restore BSEP function in all cells. Also, how AQP1 expression affects the many other canalicular solute transporters involved in bile secretion was not studied. If AQP1 action is indeed on domain redistribution rather than increased water permeability, it would be informative to study AQP1 mutants with similar effects on membrane microdomain organization, but without water transport function. Biophysical studies in cell‐culture models may be informative as well. Nevertheless, even without a convincing explanation at the mechanistic level, the normalization of bile salt secretion and serum bile salt concentration with adenoviral transfer of AQP1 is remarkable and deserves further study. So, is AQP gene therapy a credible approach for treatment of disorders of cholestasis? Given the issues discussed herein, a more transparent and plausible mechanism is needed to explain how bile flow can be increased by adenoviral delivery of AQP1 to a subset of hepatocytes. Other general concerns include differences in humans versus rats in biliary biology, particularly the ∼7‐fold lower bile flow in humans than rats per gram of liver tissue, which would confound the translation of data from experimental rat models to human disease; there are also concerns about the appropriateness of a rat estrogen model to predict therapeutic efficacy in human cholestasis. Technical challenges in gene delivery would include optimization of AQP gene delivery vectors and route for high‐efficiency transduction to safely and stably express high levels of AQPs in the canalicular membrane. Another concern is that expression of an AQP where it is not normally found is not without risk, given that transmembrane water transport and cell volume regulation are fundamental physiological processes, and AQPs are more complex than simple water conduits. If gene therapy of cholestasis is to be pursued, it would seem more logical to deliver genes encoding solute transporters/pumps to increase bile flow. Targeted pharmacological up‐regulation or activation of canalicular solute transporters by small‐molecule drugs also warrants consideration.