Physiology of the Gastrointestinal Tract || Water Transport in the Gastrointestinal Tract

1757Physiology of the Gastrointestinal Tract, Two Volume Set. DOI: © 2012 Elsevier Inc. All rights reserved.2012

10.1016/B978-0-12-382026-6.00065-8

Water Transport in the Gastrointestinal TractJay R. Thiagarajah and A.S. Verkman

65.1 INTRODUCTION

Large quantities of fluid are transported across epithelial barriers in the gastrointestinal (GI) tract for secretion of saliva, gastric juice, bile, and pancreatic fluid, and for fluid absorption in the intestine. The quantity of fluid trans-ported in the GI tract is second only to the kidney, where ~180 L of fluid per day are filtered by the glomerulus in humans and processed by various nephron segments. In the human GI tract, the salivary glands produce ~1.5 L of fluid per day, the stomach secretes 2.5 L of gastric juice, the liver produces 0.5 L of bile, the pancreas produces 1.5 L of enzyme and bicarbonate-rich fluid, the small intes-tine absorbs 6.5 L of fluid, and the colon absorbs 1.3 L of fluid against significant osmotic gradients (Figure 65.1). The fluids transported across epithelial and endothelial barriers contain salts (~150 mM) and water (~55,000 mM). As discussed elsewhere in this volume, there is a consider-able body of data on the molecular identities of the major salt transporters in epithelial cells of the GI tract and their role in transcellular ion transport. The molecular pathways for water transport in the GI tract have been identified relatively recently, and remain an evolving and, in some cases, controversial subject. As in other organ systems, the general paradigm in the GI tract is that water move-ment occurs secondary to osmotic driving forces created by active salt transport and to hydrostatic pressure dif-ferences. Based on a substantial body of evidence in the kidney and other epithelia carrying out active near-isos-molar fluid secretion or absorption, greater cell membrane water permeability produces greater net fluid movement. A description of current concepts in fluid transporting mechanisms is described in the following section.

Aquaporin (AQP) water channels provide the molecu-lar pathway for cell membrane water transport in many cell types. The AQPs are a family of small, integral mem-brane proteins that transport water and, in some cases, both water and small solutes such as glycerol. AQPs are expressed widely in cell plasma membranes in epithelial,

endothelial, and other cell types in the GI tract and else-where. As described in this chapter, there is compelling evidence for a physiological importance of some AQPs in some tissues based on studies in humans with AQP defi-ciency/mutations and phenotype analysis of transgenic mouse models of AQP deletion. However, the role of AQPs in the GI tract remains largely uncertain despite a considerable body of data on the expression pattern, cel-lular processing, and regulation of AQPs in various GI cell types. The available data are reviewed in this chapter, and major unresolved questions are identified.

65.2 EPITHELIAL FLUID TRANSPORTING MECHANISMS

65.2.1 Introduction

The mechanism of how water is absorbed and secreted across epithelia has puzzled physiologists for decades. Although large volumes of water cross the various epi-thelia of the GI tract as diagrammed in Figure 65.1, there remains a lack of consensus about the mechanisms by which water is transported across epithelia both generally and in specific regions of the GI tract. However, the gen-eral paradigm of water transport following active solute movement has remained the basis of most models of fluid transport.

The original observation that fluid can be transported in the apparent absence of an osmotic gradient was made in a series of elegant studies by a few pioneering physiologists in the late nineteenth century.1,2 It was demonstrated that fluid could be transported across an epithelial sheet such as rab-bit ileum in the absence of an external osmotic pressure dif-ference, and that this transport occurred only as long as the tissue remained viable, implying that “active” metabolism within the tissue is required. In the middle of the twentieth century Ussing demonstrated that sodium is actively trans-ported across epithelia, providing the basis for a model of fluid transport.3,4 Curran5 was the first to show that water

Chapter 65

http://dx.doi.org/10.1016/B978-0-12-382026-6.00065-8

SECTION | V Digestion and Absorption1758

transport was linearly related to Na transport, and he and Macintosh6 thereafter proposed a “three-compartment model” (Figure 65.2A) of fluid transport that accounted for the ability of epithelia to transport water in the absence or against (“uphill”) an osmotic gradient. The model requires active solute transport from compartments I to II across membrane barrier A. Compartment II is then hypertonic to compartment I. If the reflection coefficient for the actively transported solute on membrane A is greater than that on membrane B, fluid will be transferred from compartments I to III in the absence of an osmotic pressure difference between compartments I and III.

65.2.2 “Standing Gradient” Model

Following the anatomical observations of Diamond7 and Whitlock and Wheeler,8 Diamond and Bossert9,10 extended the Curran/Macintosh model by delineating the biological structures responsible for the different compartments and membranes within the epithelium, resulting in the stand-ing gradient model (Figure 65.2B). The model suggested that the lateral intercellular space (LIS) between adjacent epithelial cells was the central hypertonic compartment in the Curran-Macintosh model. The basic feature of the model, as illustrated in Figure 65.2B, is that the driving solute (Na in most cases) is actively transported from the lumen into the LIS, resulting in a steady-state (standing) osmotic gradient decreasing from the tight junction to the basolateral membrane. The osmotic gradient drives water flux through the cells into the LIS and across the basola-teral membrane, resulting in an isosmolar or near-isosmo-lar absorbate.

As originally conceived, the model predicted a number of important characteristics for an epithelium to absorb water in the absence of an osmotic gradient between the luminal and basolateral solutions, which include a water-impermeable tight junction between cells, relatively low

water permeability for membranes facing the LIS, and clustering of Na/K ATPase pumps near the tight junc-tion. However, subsequent experimental data indicated that many of the original requirements of the model were incorrect, leading to revision of the original model and the development of alternative models. Hydraulic conductiv-ity (Lp) measurements of various intestinal membranes have generally shown relatively high water permeabilities for both apical and basolateral membranes11–18 This obser-vation, together with a series of optical studies of water and solute dynamics in the LIS, led to a revised model in which the osmotic gradient localized in the LIS is pre-dicted to be so small (10 mOsm) that it is essentially unmeasurable.19–21 Another tenet of the revised model is that there is a much greater magnitude of water transport through the cell than through tight junctions.

The validity of the revised standing gradient model has remained uncertain, with an increasing number of theoreti-cal inconsistencies and problems related to the difficulty in measuring many of the key parameters in physiologi-cally relevant epithelial cells in vitro or in vivo. Reported water permeabilities of apical and basolateral membranes have differed over orders of magnitude, introducing con-siderable uncertainty about osmotic gradients required to drive fluid absorption.11,17,22 Also, the methodologies used to measure membrane water permeability have been criti-cized as has the physiological relevance of the model sys-tems.18 The high water permeabilities measured by Spring and co-workers in Necturus gallbladder and other sys-tems12,23,24 form the basis for predicting a small osmotic gradients in leaky epithelia. Earlier reported values of low water permeability have been criticized because of neglect of unstirred layer effects, although the validity of this criti-cism has also been questioned.17,25 Also, the ultrastruc-tural techniques used to identify the putative Curran and Macintosh hypertonic middle compartment as the LIS have been criticized.26–28

+1.5 L

+2.5 L

+0.5 L

+1.5 L

–6.5 L

–1.3 L

0.2 L

+2 L

Salivarygland Stomach

Liver

Gall-bladder

Smallintestine

Pancreas

Colon

FIGURE 65.1 Fluid absorption and secretion in the GI tract. Schematic of the GI tract showing daily fluid secretion and absorption in humans.

(A) (B)

(C) (D)

(E) (F)

(G) (H)

Basementmembrane

Water & Na+

Capillary

Isotonic absorbate

Na+

Na+Na+

Na+

WaterGlucose

SGLT-1

Na+

Hypertonicvillus tip

Countercurrentexchange

Epithelium

Water Na+

OSMOTIC HYDROSTATIC

A B

Isotonic

I II III

Hypertonic Isotonic

Isotonic absorbate

Water and Na+Osmosensor

Na+

Cl–

WaterNa+

_

+

Electro-osmosis

Transcellular

Paracellular

Tight junction

Aquaporins

FIGURE 65.2 Mechanisms of water transport in epithelia. (A) Curran/Macintosh three-compartment model of fluid transport. Isotonic trans-port of fluid is achieved from compartment I to III via hypertonic compartment II across semipermeable membranes A and B as a result of osmotic and hydrostatic pressures developed because of active ion transport from compartment I to II. (B) “Standing gradient” model of fluid transport. Na is actively transported into the LIS between cells resulting in transcellular movement of water into the LIS and isotonic transfer of fluid into the capillary circulation. (C) Routes of water flux across epithelia — transcellular across the lipid plasma membrane, paracellular across tight junctions between cells, transcellular via AQP channel proteins in the plasma membrane. (D) “Solute recirculation” model of fluid transport. Na is actively transported into the LIS resulting in hypertonic transfer of fluid across the basement membrane and paracellular water flux. Na is transported back into the LIS via basolateral transporters resulting in net isotonic fluid absorption. (E) Water cotransport via the sodium/glucose cotransporter SGLT-1. Water is trans-ported across the lipid bilayer along with Na and glucose as a result of conformational changes during the normal transport cycle of SGLT-1. (F) Electro-osmosis. Recirculation of Na and active transport of Cl results in an electrical current across the epithelium. Water is driven across the tight junction by electro-osmotic coupling with Na. (G) Osmosensor feedback. Na and water are transported across epithelial cells (Na water). Concurrently, water and sodium are transported across the tight junction (water Na). An osmosensor maintains absorbate isotonicity by altering the rate of tight junctional fluid transfer. (H) “Countercurrent” multiplier in small intestinal villi. Blood flow through the villous capillary network results in exchange of small solutes in the villous interstitium. Active Na absorption in the epithelium along with countercurrent exchange creates a osmolarity gradient from villous tip to base. High villous tip osmolarity drives water transport across the epithelium into the central villus lacteals.


Notwithstanding these criticisms, the revised standing gradient model of fluid movement by local osmotic gradi-ents still remains the most widely accepted description of water transport across intestinal epithelia.

65.2.3 Transcellular versus Paracellular Fluid Transport

Debate on the mechanism of solute-driven water transport in near-isotonic fluid transport in leaky epithelia (small intestine and gallbladder in vertebrate GI tract) has largely focused on whether water passes through cells (transcel-lular) or between cells (paracellular). The revised standing gradient model assumes that water movement is predomi-nantly transcellular.19 Other models based on fluid transport secondary to solute transfer, where fluid transfer is largely paracellular, include the osmosensor feedback mechanism proposed by Hill and co-workers,29 and the electro-osmotic model proposed by Fischbarg and co-workers.30 A com-pletely different, non-osmotic mechanism in which water is cotransported within solute transporters has been proposed by Zeuthen et al.31,32 Central to the debate on the pathway of water transfer across the GI tract is the role of AQPs in fluid absorption and secretion. As discussed next, stud-ies of AQP knockout mice have, in certain organs systems such as kidney proximal tubule, clarified the contribution of the transcellular pathway. In systems such as the small and large intestine where no role has yet been found for AQPs in physiological fluid movement, there is ongoing debate about the mechanism(s) responsible for fluid absorption and secre-tion (Figure 65.2C).

65.2.4 Solute Recirculation

The idea of “solute recirculation” was proposed initially in the early 1990s by Ussing et al.33 and further explored in experiments in toad intestine.34 Subsequent studies con-firmed and extended the initial experimental data, lead-ing to a mathematical model of fluid absorption in which sodium recirculation accounts for absorbate isotonicity.35 In the proposed scheme (Figure 65.2D) sodium entering into the LIS from pumping through cells or across the tight junction results in water movement. A hypertonic fluid is then extruded across the basolateral membrane of the LIS, providing the vectorial water movement component for fluid absorption. Isotonicity of the resulting absorbate is created by re-uptake of sodium, putatively by the Na/K/2Cl cotransporter, into cells and consequent “recircu-lation” back into the LIS.

The compartmental model of Larsen et al.35 used their data from toad intestine along with other experimental data to model transepithelial solute fluxes, initially for electrone-utral absorption, and subsequently for electrogenic absorp-tion.36 Their conclusions of interepithelial water flux and

basolateral Na recirculation were dependent on experi-ments using cesium as a paracellular probe to estimate the magnitude of solvent drag in the LIS and the movement of solute from the basolateral compartment. However, the use of cesium as a paracellular tracer has been criticized,37 so that definitive testing of the recirculation hypothesis remains to be done. The conflicting evidence from experimental studies and modeling24,36 about whether solute movement in the LIS represents true solvent drag resulting from water flux across the tight junction, or pseudosolvent drag result-ing from solute “swept” away by water flux through the cell, emphasizes the need for further testing of solute/solvent dynamics across epithelia.

65.2.5 Water “Pumps”

An interesting hypothesis by Wright et al.31,38–41 proposes that in the small intestine (and gallbladder) water transport is mediated by “active” transport by cotransporters such as the sodium/glucose cotransporter (SGLT-1) present at the brush border membrane of enterocytes. The theory pro-poses that water is cotransported stoichiometrically along with glucose and sodium as a consequence of conforma-tional changes that occur during the normal transport cycle of SGLT-1 (Figure 65.2E).

The principal evidence for active water transport comes from studies in Xenopus oocytes expressing SGLT-1, in which sodium current generated by the cotransporter is measured by microelectrodes, and cell swelling (a proxy for water transport across the membrane) is measured opti-cally.31,40 Oocytes overexpressing SGLT-1 swell rapidly and manifest parallel inward sodium current when exposed to an isotonic glucose-containing solution, which is inhib-ited by the specific blocker phlorizin. The initial fast rate of cell swelling was interpreted as the result of direct cou-pling of sodium and glucose transport to water transport.38 Additional evidence in support of this interpretation was the relatively slow rate of swelling produced by equivalent inward sodium currents generated by ion channels or iono-phores.38–40,42,43 Also, it was shown that rapid changes in Na transport driven by applied voltage changes induced concomitant changes in the rate of cell swelling.38 SGLT-1 has an intrinsic passive water permeability, and in the steady-state sodium glucose cotransport is expected to induce osmotic water flow by changing intracellular osmo-larity. It was proposed that in the steady state that direct (non-osmotic) water cotransport contributes ~35% of the total water transport across the membrane.39 Follow-up work by Zeuthen and co-workers has implicated active water transport by a variety of cotransporters, including the Na/glutamate cotransporter in the nervous system,44 H/lactate cotransporter in retinal pigment epithelium,45 and Na/dicarboxylate cotransporter in kidney.46 The evi-dence on which the SGLT-1 water cotransport hypothesis

Chapter | 65 Water Transport in the Gastrointestinal Tract 1761

is based has been criticized from a number of standpoints. The physiological relevance of SGLT-1 water cotransport has been questioned based on studies of water transport in the kidney. Water permeability and fluid absorption in the kidney proximal tubule are largely dependent on AQP1 as shown from knockout mouse studies (see the following section).47,48 Also, according to the proposed stoichiom-etry of coupling of water to sodium and glucose (210:2:1, hSGLT-1), the measured quantities of sodium and glucose reabsorbed by the kidney cannot account quantitatively for the quantity of water reabsorbed. 49,50 While it seems likely that cotransporter-mediated active water transport is not important in the kidney, in the small intestine this is far from clear. AQP-dependent water transport in small intestine has not been demonstrated (see the following sec-tion), so in the small intestine at least, the physiological relevance of water cotransport remains an open question.

Studies by Lapointe and co-workers51–53 called into question the interpretation of the experimental data and in some cases the accuracy of the data in studies that aimed to replicate the experimental models used by Wright et al. They did not find good agreement between membrane volt-age-induced changes in sodium current and cell swelling, concluding negligible water cotransport in the steady state. They proposed that the phenomena seen in the oocyte exper-iments can be accounted for quantitatively by the generation of local osmotic gradients close to the membrane (in effect, an unstirred layer), and concluded that the existing model of passive water flow across membranes produced by compart-mentalized osmotic gradients can explain water transport in intestinal epithelia. Thus, there remains significant debate on details of the experimental evidence supporting the hypoth-esis of water cotransport. Although water cotransport may emerge as a water transport mechanism in some epithelia under some conditions, at present it is neither sufficiently proven nor accepted to replace the established paradigm of water transport secondary to local osmotic gradients.

65.2.6 Electro-osmosis

An alternative hypothesis, in which water is transported largely via a paracellular pathway, has been proposed by Fischbarg and co-workers.30,54 Although most of the experi-mental work that underlies the model has been carried out in the cornea, proponents of the model have suggested that it is widely applicable to fluid transporting epithelia based on previous studies showing current-induced fluid move-ment.25,33,55 The observation that underlies the model is that current across an epithelium results in fluid flows that mir-ror the direction and magnitude of the electrical current.30 In the model, active ion transport and recirculation gener-ate a circulating current and transepithelial potential differ-ence. The solute counterion is concurrently driven across the tight junctions between the epithelial cells. Fluid flow across

the tight junctions is consequently generated by the electro-osmotic coupling of water and the solute counterion, result-ing in paracellular fluid transport and isotonic fluid transport across the epithelium, although the latter is dependent on the exact coupling ratio between water and counter ion (Figure 65.2F). The experimental basis of this theory has largely hinged on a series of studies showing current-induced fluid flow from the corneal stroma across the corneal endothe-lium in isolated, in vitro rabbit corneal preparations.30,54,56,57 In addition, a computational model of the corneal endothe-lium was developed to predict solute and fluid transport responses under experimental conditions.58

Although the idea of electro-osmosis has been around for many years, its application to epithelial physiology, both theoretically and experimentally, has been a relatively recent phenomenon. In general, the theory seeks to resolve some of the long-standing experimental inconsistencies in local osmosis models of fluid transport. A key discrepancy highlighted by the proponents of the theory is the quanti-tative difference between the reduction in osmotic water permeability (40–50%) and the reduction in fluid transport (20%) in certain leaky epithelia in AQP knockout mouse models.57 Theoretical objections to electro-osmosis have centered on the explanation that ion flow also produces solute concentration gradients close to the membrane, and current-induced water flow is therefore governed by local osmosis. The main counter to this argument is the fact that some experimental data show that the onset of fluid flow generated by changing transepithelial current is very fast (3 s) and therefore too fast to be explained by establish-ment of a local solute concentration gradient. Another the-oretical issue stems from known fluid flows generated by non-ionic, osmotically active impermeant solutes, which are not quantitatively explained by electro-osmosis alone. Lastly, the theoretical treatment of electro-osmosis is cru-cially dependent on the number of parameters that only have been tested in the cornea or are not fully described in the model. These include most crucially the coupling ratio at the tight junction, but also the composition of the tight-junction charges and hydrostatic pressure effects within different epithelial compartments.

The electro-osmosis model represents an interesting departure from the largely transcellular models of fluid transport, and highlights the largely underappreciated importance of the tight junction in transepithelial water transport. However, the electro-osmosis model has not been validated experimentally in different epithelia nor have its predictions been quantitatively confirmed.

65.2.7 Osmosensor Feedback Model

Hill and co-workers29 proposed an interesting solution to the problem of isotonicity of transported fluid in epithelia, which relies on paracellular water flow and an osmosensor


(Figure 65.2G). The basis of the model is that fluid trans-port across an epithelium is a blend of transcellular and paracellular solute and water transport. Water is trans-ported across the cell osmotically and is therefore largely hypertonic. Paracellular water flow is generated by forced convection of fluid through tight junctional channels that reflect solute over water, resulting in a largely hypotonic fluid. The magnitude and rate of this junctional fluid flow is proposed to be controlled by an osmosensor that sam-ples the source solution. The fluid transported across the epithelia is osmotically clamped by the osmosensor via regulation of the rate of fluid traversing the tight junctions, resulting in a transported solution with a tonicity close to that of the source solution.

The osmosensor model as proposed relies on experimen-tal data showing significant junctional fluid flow using dex-trans as probe molecules.59–61 However, besides this indirect experimental evidence on paracellular fluid flow, the model remains speculative. AQPs were proposed to be the candidate osmosensor crucial to the model, although the mechanism by which they function in this capacity has not been described, and there is no experimental evidence to support this con-jecture. Further, the cellular signaling mechanisms and tight junctional apparatus involved in controlling junctional fluid transfer have not been experimentally described. Therefore, although an interesting idea to explain fluid transport in the absence of an osmotic gradient between source and sink solutions, the osmosensor model is at present a speculative, unsubstantiated theory.

65.2.8 Other Transport Theories

65.2.8.1 Countercurrent Multiplication

An intriguing model of fluid absorption, involving coun-tercurrent exchange in the small intestinal villus, was pro-posed by Lundgren and co-workers about 40 years ago.62 The idea that a countercurrent system operates in the vil-lus was based initially on observations on the vascular morphology of mammalian villi.63–65 The villus contains a central arterial vessel surrounded by a dense capillary net-work and neighboring venous vessels. The hairpin arrange-ment of the arterial and venous vessels, resembling the renal microvasculature, could produce a “countercurrent” flow system in which blood flow occurs in opposite direc-tions. The evidence that this countercurrent flow could operate as an exchange system came from measurements of relative absorption rates of the inert gases 133Xe and 85Kr from the intestinal lumen into the villus.66,67 These experiments, along with measurements of villous hemody-namics, suggested the presence of a villous countercurrent exchanger in several species including man.68

These observations led to the proposal that the puta-tive villous countercurrent exchanger could function as

an osmotic “multiplier” as in the renal medulla.69 The evidence to support a multiplier function comes mainly from measurements of villous osmolality using cryoscopic techniques and sodium-sensitive microelectrodes.70–73 The villus was reported to have a large gradient of osmola-lity from tip to base with tip osmolality as high as 700–900 mOsm. From these measurements it was proposed that the villous interstitium was the hypertonic compartment described in the Curran-Macintosh model. Water absorp-tion from the intestinal lumen could be driven by the large osmotic gradient/pressure head developed in the villous tip by the countercurrent multiplier system (Figure 65.2H).

The theoretical basis of countercurrent exchange and its presence in intestinal villi has been criticized (for more theoretical details see 74). The accuracy and validity of the techniques used to measure inert gas absorption to dem-onstrate countercurrent exchange have been criticized and alternative explanations for the relative gas absorption rates have been proposed.75,76 Conflicting data has also emerged on the existence of countercurrent exchange in certain mam-malian species but not others, based on differences in vil-lous anatomy.77 The in vitro techniques used to show high villous tip osmolality are thought to produce highly vari-able results, which may be an artifact of the tissue prepara-tion. It has been pointed out that the microvascular anatomy in small intestinal villi is quite different from that of the renal medulla, and in particular there is no equivalent path-way in villi to the ascending limb of Henle in the kidney.76 Also it has been argued that the existence of a hypertonic compartment in the villous tip does not necessarily require a countercurrent mechanism but does require a relatively water-impermeable epithelium.

Thus, convincing in vivo evidence for a hypertonic villous compartment is lacking. Modern optical and fluo-rescent techniques may be able to elucidate the solute/solvent dynamics within the villus, although experiments on the small intestine in situ remain a significant techni-cal challenge due to its complex geometry. Additionally, the efficiency of the putative countercurrent exchange sys-tem is critically dependent on the water permeability of the microvasculature, suggesting that endothelial AQP1 might play an important role in villous fluid absorption.

65.2.8.2 Luminal Hypotonicity Driven by Acidification

An interesting model for fluid absorption in small intestine was proposed by Lucas78 in which the driving force for water movement across the small intestinal mucosa is located at the brush border membrane of enterocytes. This model pos-tulates that fluid transport is driven by the neutralization of bicarbonate ions contained within an unstirred layer at the luminal membrane of enterocytes. Sodium–proton exchange at the brush border membrane produces local luminal


acidification resulting from proton transport out of the cell. Bicarbonate ions diffusing from the bulk solution neutralize the protons, resulting in the production of water and carbon dioxide. The combination of two osmotically active ions to form a single osmotically active carbon dioxide molecule produces a hypotonic compartment adjacent to the luminal membrane, which, together with an increase in intracellular osmolarity from sodium entry and carbon dioxide diffusion into the cell, drives water across the membrane. Evidence for this model comes from observations of increased fluid absorption for bicarbonate-containing balanced electrolyte solutions in the upper small intestine where luminal acidifi-cation is high, along with data from bacterial diarrheas.79–85 In Salmonella typhimurium infection, it was shown that the ileum, which normally exhibits robust electrogenic Na/glucose absorption, exhibits jejunum-like characteristics with increased luminal acidification and elevated plasma pCO2. This was proposed as evidence of increased Na/H exchange leading to fluid absorption.

Although the model suggests that the brush border mem-brane is the key site of solute–solvent coupling, it does not ultimately contradict the original Curran/Macintosh idea that a basolateral or abluminal hypertonic compart-ment drives fluid absorption. In the case of electrogenic sodium absorption by sodium channels or sodium/glucose cotransport, or absorption in the absence of luminal acidi-fication, sodium movement across the epithelium into a putative hypertonic compartment remains the primary driv-ing force for fluid movement. In addition the model does not adequately explain how an osmotic gradient across the apical membrane alone results in overall fluid trans-port from lumen to blood, and recent studies of bacterially induced fluid transport argue against a significant role.86 Notwithstanding these caveats, the concept of a luminal hypotonic compartment potentially describes a number of unexplained observations in the upper small intestine. It remains unlikely, however, that it can describe fluid trans-port in other parts of the GI tract where luminal acidification does not normally occur.

65.2.9 Summary

In summary, although the general paradigm of local osmotic and hydrostatic pressure differences driving fluid transport across epithelia has been sufficient to describe measured water flows in the GI tract, a number of variations of the basic theory have been proposed as well as some completely new theories. The mechanisms involved in fluid absorp-tion have, in general, received much more attention than the mechanisms of fluid secretion particularly in complex epi-thelia such as the small intestine. Secretion of saliva, bile, and pancreatic fluid can probably be explained simply and adequately by water movement into a closed/slowly mov-ing compartment driven by osmotic gradients produced by

active salt pumping. However, in small and large intestine and in the gallbladder there remains significant debate on the basic water/fluid-transporting mechanisms.

65.3 AQPs

65.3.1 Introduction

There is an extensive body of information on the tissue localization, regulation, structure, and function of the more than 10 mammalian AQPs identified. Structural studies of AQP1 indicate a homotetrameric assembly in membranes in which each monomer contains six tilted helical segments that form a barrel surrounding a water pore (reviewed in 87). Functional measurements indicate that AQPs 1, 2, 4, 5, and 8 are primarily water selective, whereas AQPs 3, 7, and 9 (“aquaglyceroporins”) also transport glycerol and possibly other small solutes. Structural analysis and molecular mod-eling suggest that AQP water selectivity is a consequence of steric and electrostatic factors in which water moves through a narrow pore in a tortuous, single-file manner that does not permit continuous hydrogen bonding.88 The AQPs are expressed in many cell types involved in rapid fluid trans-port such as kidney tubules, exocrine gland epithelia, micro-vascular endothelia, choroid plexus, and ciliary epithelium. Physiological roles of AQPs have been proposed based on their tissue expression pattern and in some cases the exist-ence of human mutations, such as nephrogenic diabetes insipidus caused by AQP2 mutation;89–91 however, the una-vailability of AQP inhibitors suitable for use in vivo has pre-cluded direct investigation of their function. As described in the following section, phenotype analysis of AQP knockout mice has elucidated specific physiological roles for many of the AQPs and has established general paradigms for their physiological roles.

65.3.2 Physiological Roles of AQPs in Non-gastrointestinal Tissues

65.3.2.1 AQPs in the Kidney Tubules and the Urinary Concentrating Mechanism

The role of AQPs in kidney function has been studied extensively (reviewed in 92). AQP1 is expressed in apical and basolateral plasma membranes in proximal tubule and thin descending limb of Henle as well as in microvascular endothelia of outer medullary descending vasa recta. AQP2 is expressed in collecting duct principal cells and undergoes antidiuretic hormone (vasopressin)-regulated trafficking between an intracellular vesicular compartment and the cell apical plasma membrane. AQP3 and AQP4 are expressed at the basolateral membrane of collecting duct epithelial cells, with relatively more AQP3 in proximal segments of col-lecting duct and more AQP4 in inner medullary collecting


duct. Each of these AQPs plays a role in the formation of a concentrated urine. Given free access to fluid, AQP1- and AQP3-null mice were remarkably polyuric, consuming 2–3 (AQP1) to 7–10 (AQP3) times more fluid than wild-type mice, whereas the AQP4-null mice were not polyuric and manifested only a mild reduction in maximal urinary con-centrating ability.93,94 After a 36 hour water deprivation, urine osmolality in AQP1-null mice did not change, whereas that in AQP3-null mice increased to a submaximal level. Mutant AQP2 mice, in which a mutation causing nephro-genic diabetes insipidus in human subjects was introduced into the mouse genome, were severely polyuric and died within the first 10 days after birth.95 Mechanistic studies indicated that the urinary-concentrating defect in AQP1-null mice results from defective proximal tubule fluid absorp-tion and defective countercurrent multiplication produc-ing a relatively hypo-osmolar medullary interstitium.47,96,97 Humans with AQP1 deficiency manifest a qualitatively similar urinary-concentrating defect,98 although maxi-mum urine osmolality in humans (~1200 mOsm) is much lower than that in mice (3000 mOsm). Defective urinary-concentrating ability in AQP2, AQP3, and AQP4 deficiency result from reduced collecting-duct water permeability and consequent impaired osmotic equilibration of tubular fluid with the hypertonic medullary interstitium.

65.3.2.2 AQPs in Near-isosmolar Fluid Absorption and Secretion

As mentioned previously, impaired fluid absorption in kid-ney proximal tubule in AQP1 deficiency indicates the need for high, transepithelial water permeability for rapid, near-isosmolar fluid transport.47 Indeed, micropuncture studies showed remarkable luminal hypertonicity in late proximal tubule in AQP1-null mice resulting from the retrieval of a hypertonic absorbate.48 An important role for AQPs was also found for near-isosmolar fluid secretion in salivary99 and airway submucosal100 gland in AQP5 deficiency, and for AQP1-dependent secretion of cerebrospinal fluid by choroid plexus101 and aqueous fluid by the ciliary epi-thelium.102 For example, saliva secretion was reduced by more than twofold in AQP5-deficient mice, and secreted saliva was hypertonic as a consequence of active salt secre-tion in the acinar lumen of salivary gland without adequate osmotic equilibration because of reduced epithelial water permeability. High, transepithelial water permeability per-mits rapid water transport in response to active transepithe-lial salt transport. However, active fluid transport in many tissues does not appear to be AQP dependent, such as alve-olar fluid absorption in AQP1/AQP5 deficiency103,104 and sweat secretion in AQP5 deficiency.105 The rate of active fluid absorption/secretion per unit epithelial surface area in alveolus and sweat gland is much lower than that in kidney proximal tubule or salivary gland, suggesting that

high, AQP-dependent water permeability is not required to sustain relatively slow fluid absorption/secretion. A simi-lar conclusion was obtained in gallbladder, where AQP1 was found to increase transepithelial osmotic water perme-ability by 10-fold, but not affect physiologically important gallbladder functions such as bile concentration.106

65.3.2.3 AQPs in Brain Swelling and Neural Signal Transduction

AQP4 has interesting roles in the CNS, where it is expressed strongly throughout the brain and spinal cord, especially in astroglial cells lining ependyma and pial sur-faces in contact with the CSF and the blood–brain barrier. Although AQP4-null mice show no overt neurological abnormalities, they had remarkably reduced brain swelling after cytotoxic (cellular) edema produced by acute water intoxication and ischemic stroke.107,108 However, brain swelling, intracranial pressure, and clinical outcome were worse in AQP4-null mice in models of vasogenic (leaky vessel) edema including intraparenchymal fluid infusion, cortical freeze injury, and brain tumor.109 Since AQP4 facil-itates bidirectional water transport, its deletion in mice can thus reduce brain water accumulation in cytotoxic edema, resulting in improved clinical outcome, and reduced clear-ance of excess brain water in vasogenic edema, worsening clinical outcome.

Interestingly, AQP4 is also expressed in CNS, inner ear, and retina in supportive cells that are in close proximity to non-AQP-expressing electrically excitable cells — in astro-glia supporting neurons, in retinal Müller cells supporting bipolar cells, and in cochlear Clausius and Hensen’s cells supporting hair cells. Mice lacking AQP4 showed reduced seizure susceptibility,110 and reduced evoked potential responses to light,111 acoustic,112 and olfactory113 stimuli. Also, mice lacking AQP4 showed remarkable retinal neu-roprotection after ischemic–reperfusion challenge.114 Based on AQP4 and K channel Kir4.1 colocalization, it has been proposed that AQP4 in supportive cells may facilitate K recycling during electrical signal transduction in brain, retina, and inner ear.115 Although AQP4 does not directly modulate the activity of Kir4.1,116 there may be alterations in extracellular space dynamics in AQP4 deficiency as a consequence of salt/water coupling. In support of this possi-bility, we found, using microfiberoptic fluorescence photob-leaching and ratio imaging methods, that the extracellular space was expanded in cerebral cortex in AQP4-deficient mice.117,118 Quantitative studies relating extracellular space volume and ionic concentration to neural stimuli are needed to establish the mechanism of altered neural signal trans-duction in AQP4 deficiency. In the GI tract, AQP1 has been localized to glial cells in mesenteric and pancreatic nerve ganglia,119 although its function in the peripheral nervous system remains unknown.


65.3.2.4 AQPs in Corneal Edema and Transparency

Studies of corneal swelling suggest yet another interest-ing role for AQPs. Maintenance of corneal transparency requires precise regulation of stromal water content. AQP1 is expressed in corneal endothelial cells and AQP5 in epi-thelial cells. Corneal thickness, water permeability, and response to experimental swelling were measured in wild-type mice and mice lacking AQP1 or AQP5.120 Compared to wild-type mice with corneal thickness of 123 µm, cor-neal thickness was reduced in AQP1-null mice (101 µm) and increased in AQP5-null mice (144 µm). After expo-sure of the external corneal surface to hypotonic saline (100 mOsm), the rate of corneal swelling (5 µm/min) was reduced approximately twofold by AQP5 deletion. After exposure of the corneal endothelial surface to hypotonic saline by anterior chamber perfusion, the rate of corneal swelling (7 µm/min) was reduced approximately fourfold by AQP1 deletion. Although baseline corneal transparency was not impaired by AQP1 deletion, the recovery of cor-neal transparency and thickness after hypotonic swelling (10 minutes exposure of corneal surface to distilled water) was remarkably delayed in AQP1-null mice, with ~75% recovery at 7 minutes in wild-type mice compared to 5% recovery in AQP1-null mice. The impaired recovery of corneal transparency in AQP1-null mice provides evidence for the involvement of AQP1 in active extrusion of fluid from the corneal stroma across the corneal endothelium.

65.3.2.5 Roles of Aquaglyceroporins — AQP3 in Skin Hydration and Cell Proliferation and AQP7 in Fat Cells

The water/glycerol transporting protein AQP3 is expressed strongly in epidermal keratinocytes, which form the inter-face between the dermis/vasculature and the stratum corneum. The hypothesis that AQP3 is an important deter-minant of skin hydration and barrier/mechanical proper-ties was tested by comparative measurements in wild-type and AQP3-null mice generated in a hairless genetic back-ground.121 High-frequency skin conductance, a direct measure of stratum corneum hydration, was remarkably (approximately twofold) reduced in the AQP3-null mice, and this difference persisted even when mice were placed in a humidified atmosphere or when the skin was covered by an occlusive dressing. Transepidermal water loss meas-urements after tape-stripping revealed significantly slowed recovery of barrier function in the AQP3-null mice. Skin elasticity, measured from the kinetics of skin displacement in response to rapidly applied suction, was remarkably reduced in the AQP3-null mice due to abnormal stratum corneum mechanical properties; skin elasticity of wild-type and AQP3-null mice became similar after removal of

the stratum corneum by tape-stripping. AQP3 thus plays an important role in skin hydration and barrier/mechani-cal properties. A detailed comparison of skin morphology and the composition of the stratum corneum and epidermis revealed reduced glycerol content as the main abnormality in AQP3 deficiency.122 The various abnormalities in stra-tum corneum hydration, barrier recovery, and skin elastic-ity could be related to reduced glycerol content based on the humectant (water-retaining) and biosynthetic functions of AQP3. In addition, topical or systemic glycerol replace-ment corrected the skin abnormalities in the AQP3-null mice,123 supporting the conclusion that the glycerol-trans-porting function of AQP3 is important for skin hydration and biosynthetic functions.

In addition to epidermal keratinocytes, AQP3 is also expressed in corneal and colonic epithelium. We discov-ered a novel role for AQP3 in cell proliferation in studies of wound healing124 and tumorigenesis125 in skin, corneal sur-face repair,126 and colonic regeneration.127 AQP deficiency was associated with slowed healing of cutaneous and cor-neal wounds, with reduced cell proliferation as measured by BrdU incorporation. Motivated by these results supporting the involvement of AQP3 in cell proliferation, we discov-ered a remarkable phenotype using a skin tumor model of epidermal cell proliferation in which cutaneous papillomas were produced by repeated inducer–promoter exposure.125 Mice lacking AQP3 failed to produce papillomas following the same treatment in which all wild-type mice produced multiple papillomas. Studies in various intact skin and keratinocyte culture models suggested that AQP3-facilitated glycerol transport is a key determinant of cell proliferation by a mechanism involving reduced epidermal glycerol con-centration in AQP3 deficiency, resulting in impaired lipid biosynthesis and reduced glycerol metabolism and ATP, with consequent impairment in MAP kinase signaling and reduced cell proliferation. AQP3-dependent cell prolifera-tion probably is relevant to epithelial regeneration in colitis, as discussed in the following section, and for the growth and proliferation of some GI tumors.

AQP7, which is expressed strongly in adipocyte plasma membranes, also functions as a water/glycerol transport-ing protein. We found remarkable age-dependent adipocyte hypertrophy in AQP7-deficient mice and attributed the pheno-type to defective AQP7-dependent glycerol transport.128 Wild-type and AQP7-null mice had similar growth at 0–16 weeks as assessed by body weight, although by 16 weeks AQP7-null mice had 3.7-fold increased body fat mass. Adipocytes from AQP7-null mice of age 16 weeks were greatly enlarged (diameter 39 µm) compared to wild-type mice (118 µm). Adipocytes from AQP7-null mice also accumulated excess glycerol (251 vs. 86 nmol/mg protein) and triglycerides (3.4 vs. 1.7 µmol/mg protein). In contrast, at age 4 weeks, adi-pocyte volume and body fat mass were comparable in wild-type and AQP7-null mice. To investigate the mechanism(s)


responsible for the progressive adipocyte hypertrophy, glyc-erol permeability and fat metabolism were studied in adi-pocytes isolated from the younger mice. Plasma membrane glycerol permeability measured by 14C-glycerol uptake was threefold reduced in AQP7-deficient adipocytes. However, adipocyte lipolysis, measured by free fatty acid release and hormone-sensitive lipase activity, and lipogenesis, measured by 14C-glucose incorporation into triglycerides, were not affected by AQP7 deletion. The results supported the conclu-sion that adipocyte hypertrophy in AQP7 deficiency results from defective glycerol exit and consequent accumulation of glycerol and triglycerides, suggesting the possibility of pharmacological upregulation of adipocyte AQP7 expres-sion/function to reduce adipocyte size and fat mass in obesity (reviewed in 129).

65.3.2.6 AQPs in Angiogenesis and Cell Migration

We discovered a new function of AQPs in facilitating cell migration.130 The original motivation for studying the role of AQPs in angiogenesis was the observation of strong AQP1 expression in tumor microvessels. Remarkably, impaired tumor growth was found in AQP1-null mice after subcutaneous or intracranial tumor cell implantation, with reduced tumor vascularity and extensive necrosis. Reduced microvessel growth was also found in implanted pellets of Matrigel containing vascular endothelial growth fac-tor. A novel mechanism for the impaired angiogenesis was established from cell culture studies. Although adhesion and proliferation were similar in primary cultures of aor-tic endothelia from wild-type versus AQP1-null mice, cell migration was greatly impaired in AQP1-deficient cells, with abnormal vessel formation in vitro. Stable transfec-tion of non-endothelial cells with AQP1, or a structurally different water-selective transporter (AQP4), accelerated cell migration and in vitro wound healing. Interestingly, the migrating AQP1-expressing cells had prominent mem-brane ruffles at the leading edge with polarization of AQP1 protein to lamellipodia, where rapid water fluxes occur. As a possible mechanism for the dependence of cell migra-tion on AQPs, we proposed that cell membrane protru-sions are formed as a consequence of actin cleavage and ion uptake at the tip of a lamellipodium, creating local osmotic gradients that drive the influx of water through the cell membrane.131,132 Water entry would then increase local hydrostatic pressure to cause cell membrane protru-sion, resulting in forward cell movement. The presence of an AQP in the lamellipodium would augment this process.

AQP-facilitated cell migration appears to be a gen-eral phenomenon that is not cell-type or AQP specific (reviewed in 133). For example, we have characterized AQP4-dependent cell migration in kidney proximal tubule cells,134 AQP4-facilitated migration of astroglia cells in

brain,135 and AQP3-facilitated migration of epidermal keratinocytes124 and corneal epithelial cells.126 A recent study reported circumstantial evidence that AQP1 facili-tates pathological angiogenesis in cirrhosis.136 Of particu-lar interest was the observation that AQP expression in tumor cells enhanced their migration, metastatic potential, and local invasiveness.137 Many tumors express AQPs and in some cases AQP expression correlates well with tumor grade (reviewed in 138). The enhancement of tumor inva-sion by AQPs suggests AQP inhibition as a new approach for tumor therapy.

65.3.3 General Paradigms About Physiological Functions of AQPs

In summary, studies of the extra-GI phenotype of AQP-deficient mice suggest that AQP-facilitated water perme-ability is important: when water movement is driven across a barrier by a continuous osmotic gradient (as in kidney col-lecting duct); for active, near-isosmolar fluid absorption/secretion (as in kidney proximal tubule and salivary gland); for neural signal transduction (as in brain and inner ear); and for cell migration (as in tumor vessel angiogenesis). Glycerol transport by the aquaglyceroporins is involved in skin hydration and cell proliferation (AQP3) and adipocyte metabolism (AQP7). Another conclusion from the pheno-type studies is that the tissue expression of an AQP does not ensure its physiological significance, so that evalua-tion of tissue AQP function must be done on a case-by-case basis. For example, despite the expression of AQP4 in skeletal muscle, no phenotype abnormalities in AQP4-null mice were found,139 as was the case for several AQPs in air-ways and lung.140 From these general paradigms, a variety of diverse roles of AQPs in the GI tract are possible, as dis-cussed in the following section.

65.3.4 AQP Expression in the GI Tract

The expression of specific AQPs in the GI tract provides clues to possible functional roles. Several well-documented expression patterns provide indirect evidence for a possible role of AQPs in these tissues, including: AQP1 in intrahepatic bile duct epithelium (bile formation), intestinal lacteals (fat absorption), and microvascular endothelia throughout the GI tract (fluid absorption/secretion); AQP4 in basolateral mem-brane of parietal cells in stomach (acid/fluid secretion) and colon surface epithelium (fluid absorption); AQP5 in apical membrane of acinar cells in salivary glands (saliva secre-tion); AQP8 in salivary gland, liver, pancreas, and intestine; and AQP9 in hepatocytes. Figure 65.3 shows examples of AQP protein localization in the GI tract, with AQP1 in cen-tral lacteals of small intestine (A), AQP4 at the basolateral membrane of surface epithelial cells in colon (B), AQP8 in crypt epithelium in ascending colon (C), and AQP4


at the basolateral membrane of gastric parietal cells (D). Additional information about the localization of these and other AQPs is discussed in the following section, together with information about organ-specific fluid-transporting mechanisms and available data from AQP knockout mice.

65.4 FLUID TRANSPORT MECHANISMS AND AQPS IN GI ORGANS

65.4.1 Salivary Gland

Saliva secreted by the salivary gland is the first fluid with which ingested food comes into contact. The interstitial-to-luminal transport of sodium and chloride across the acinar epithelium is the driving force for osmotic water

movement.141 The salivary duct epithelium is believed to be relatively water impermeable, in which the sodium and chloride are absorbed and potassium and bicarbonate secreted to produce a hypotonic saliva. Upon stimulation the salivary gland can secrete saliva at high rates (up to 50 ml/min/100 g tissue in humans), which relies on rapid water movement from serosa to mucosa across the capil-lary endothelium and acinar cells. The possible involve-ment of AQPs in this process has been proposed based on the expression of AQP1 in microvascular endothelial cells of salivary gland,142 AQP5 in apical membrane of acinar cells,143 and AQP8 in acinar cells.144,145 A more recent study of expression of AQPs 1, 3, 4, and 5 in human sali-vary gland reported AQP1 in microvessels and AQP5 at the apical membrane of acinar cells as found in rodent salivary

(A) (B)

(C) (D)

FIGURE 65.3 Immunocytochemistry of AQP expression in the GI tract. (A) Immunofluorescence of mouse small intestinal villi showing AQP1 protein in central lacteals. (B) AQP4 labeling of basolateral membrane colonic surface epithelium. (C) AQP8 labeling of colon crypt epithelium. (D) AQP4 labeling of basolateral membrane in gastric parietal cells. (Adapted from 148,160,172,239.)


gland, as well as AQP3 at the basolateral membrane of both serous and mucous acinar cells.146

Our laboratory generated AQP5 knockout mice to inves-tigate the role of AQP5 in saliva secretion.99 Pilocarpine-stimulated saliva production was reduced by more than 60% in AQP5 knockout mice. Compared with the saliva from wild-type mice, the saliva from knockout mice was hypertonic (420 mOsm) and dramatically more viscous (Figure 65.4). Amylase and protein secretion, functions of salivary mucous cells, were not affected by AQP5 deletion. Saliva secretion was not impaired in AQP1 or AQP4 knockout mice. A sub-sequent report confirmed the defect in saliva secretion in AQP5-null mice and reported reduced water permeability, as expected, in acinar cells isolated from the null mice.147 In fol-low-on studies we found no effect of AQP8 deletion on saliva secretion in mice,148 and there was no difference in saliva secretion in AQP8/AQP5 double knockout mice when com-pared to AQP5 knockout mice.

One study suggested that AQP5 has an abnormal intra-cellular distribution in Sjögren’s syndrome subjects,149 although another study reported the opposite finding.150 Another immunolocalization study in rat showed that AQP5 in salivary gland acinar cells is primary at the apical plasma membrane under both basal and stimulated conditions.151 Although there may be a subset of subjects with Sjögren’s syndrome with cellular mislocalization of AQP5, it is more likely that AQP5 mislocalization, if it occurs, is a conse-quence of the chronic immune damage to the salivary gland. There has be interest in the possibility of AQP gene therapy to treat hyposalivation in post-irradiation salivary gland dysfunction by transfer of AQP1 using a viral vector.152 However, the utility of this approach will await human clinical trials, as it is unclear whether water permeability

is rate-limiting in salivary gland acini surviving radiation exposure, it is uncertain if the therapy will be effective.

65.4.2 Stomach

The large quantity of gastric fluid produced by the mam-malian stomach is thought to be secreted mainly by fundic glands in the mucosa of the stomach body. These glands contain mucous cells, chief cells, and parietal cells that secrete mucus, pepsinogen, and hydrochloric acid, respec-tively. During agonist-stimulated acid secretion, gastric juice is transported from the mucosal interstitium into the human gastric lumen at a rate of ~0.7 ml/min/100 g tissue.153 There is little information about the relative contributions of dif-ferent cell types involved in gastric fluid secretion. So far, AQP4 is the only AQP identified in the stomach. Rat AQP4 was immunolocalized to the basolateral membrane of pari-etal cells.154 A human AQP4 homolog was subsequently cloned from stomach and immunolocalized to both pari-etal cells and chief cells.155 A more recent evaluation of AQP4 expression in gastric glands showed AQP4 protein localization only in parietal cells in mid and deep regions of gastric glands.156 One interesting study addressed the pos-sible importance of AQP4 assembly in orthogonal arrays of proteins (OAPs), which are lattice-like particle arrange-ments seen by freeze-fracture electron microscopy. AQP4 was shown to be the main component of OAPs from stud-ies in AQP4-expressing CHO cells157 and from the absence of OAPs in multiple tissues in AQP4-deficient mice.158 Carmosino et al.159 reported that OAPs in an AQP4-transfected human gastric cell line was reduced by ~50% after histamine stimulation, suggesting that short-term regu-lation of AQP4 assembly and water permeability might be

–/– +/– +/+

+/+

AQP5(A) (B)

AQP5 –/–

+/+

AQP5 –/– *

*

0

0 50 100 150

100 200 300 400 500

Osmolality (mOsm)

[Na+] (mM)

FIGURE 65.4 Defective salivary gland secretion in AQP5-null mice. (A) Photograph of saliva collected over 5 minutes from mice of indicated genotype. Salivation was stimulated by pilocarpine. (B) Averaged (SEM) osmolality and sodium concentration of saliva collected from mice of indi-cated genotype. (Adapted from 99.)


possible. The physiological significance of this observation is unclear. It has been postulated that AQP4 is involved in gastric acid and pepsinogen secretion and/or cell volume regulation.

One study investigated the role of AQP4 in gastric acid and fluid secretion utilizing AQP4-null mice.160 Gastric acid secretion was measured in anesthetized mice in which the stomach was lumenally perfused at 0.3 ml/min with normal saline containing 14C-PEG as a volume marker. Collected effluent was assayed for titrable acid content and 14C-PEG radioactivity. After a 45 minute baseline perfusion, acid secretion was stimulated by intravenous pentagastrin for one hour or intravenous histamine plus intralumenal carbachol. Baseline gastric acid secretion was 0.06 0.03 μEq/15 min in wild-type mice versus 0.03 0.02 μEq/15 min in AQP4-null mice. Pentagastrin-stimulated acid secre-tion was 0.59 0.14 μEq/15 min in wild-type mice versus 0.70 0.15 μEq/15 min in AQP4-null mice. Histamine/car-bachol-stimulated acid secretion was 7.0 1.9 μEq/15 min in wild-type versus 8.0 1.8 μEq/15 min in AQP4-null mice. In addition, there was no effect of AQP4 deletion on gastric fluid secretion, gastric pH, or fasting serum gastrin concentrations. Thus, there is no direct evidence to support a physiological role of AQPs in the stomach.

65.4.3 Liver

The expression of several AQPs (1, 3, 8, and 9) has been reported in liver and proposed to play a role in bile secre-tion. Early immunocytochemistry showed AQP1 expression on the apical and basolateral membranes of cholangi-ocytes.161 A series of studies by the LaRusso group provided indirect evidence for a possible role of several AQPs in bile secretion. Semi-quantitative water permeability meas-urements in isolated cholangiocytes and intrahepatic bile ducts suggested AQP-mediated water transport based on weakly temperature-dependent water transport and inhi-bition by HgCl2.

162 Membrane fractionation studies sug-gested that secretin caused a redistribution of AQP1 from intracellular vesicles to the cell plasma membrane, and that the redistribution was blocked by colchicine and low tem-perature.163,164 If correct, these observations would indicate that, unlike other cell types where AQP1 is expressed con-stitutively at the plasma membrane, the cholangiocyte pos-sesses a unique regulatory mechanism for AQP1. However, the cell biology of regulated AQP1 trafficking will require elucidation, since AQP1 (unlike AQP2165) does not con-tain a consensus phosphorylation site at its C-terminus. Immunocytochemistry in rat166,167 and mouse168 showed AQP8 protein expression in intracellular vesicles in hepa-tocytes. Further measurements of water permeability in isolated hepatocytes from rat showed moderate water per-meability with Pf of 66 104 cm/s at 37°C;169 however, follow-up studies from the same group reported a much

lower Pf of 104 cm/s.170 They also reported a cAMP ago-nist-induced approximately twofold increase in rat hepato-cyte water permeability, relocalization of intracellular AQP8 to the plasma membrane,167 and a sixfold increase in water permeability of canalicular plasma membrane domains in cAMP-treated rat hepatocytes.171 Follow-on work from our group showed AQP8 immunolocalization in the plasma membranes of hepatocytes in mice with weak intracellu-lar labeling.148 AQP8 knockout mice served as controls for immunolocalization analysis. Osmotic water permeability measured in freshly isolated hepatocytes from wild-type mice was low (Pf 6 104 cm/s) and not increased after cAMP agonists or reduced in AQP8 deficiency, provid-ing evidence against constitutive or cAMP-regulated AQP8 water permeability in hepatocytes in mice. As mentioned for AQP1, rat and mouse AQP8 do not contain consensus sequences for phosphorylation by protein kinases A or C.

Studies in AQP1 and AQP8 knockout mice do not sup-port a significant role of AQPs in bile secretion, at least in mice. Dietary fat misprocessing was observed in young but not adult AQP1 knockout mice.172 Whereas young wild-type mice gained 36 5% body weight in 8 days, the AQP1-null mice lost 2 1% body weight and developed stea-torrhea. The weights became similar 6 days after return to a 6% fat diet. Despite a 50% decrease in ad libitum food intake in the knockout mice, averaged serum triglyceride concentrations were 137 mg/dl in wild-type versus 66 mg/dl in knockout mice on the high fat-diet. Semiquantitative analysis of stool fat content by a lipid extraction method showed elevated stool fat in the knockout mice on the high-fat diet. Inclusion of pancreatic enzymes (lipase, amy-lase, and protease) in the high-fat diet partially corrected the weight loss and the increased stool fat content in the AQP1 knockout mice. Fluid collections done in older mice (which are less sensitive to a high-fat diet) by ductal can-nulation showed threefold increased pancreatic fluid flow in response to secretin/CCK, but volumes, pH, and amylase activities were affected little by AQP1 deletion. Bile flow rates and bile salt concentrations were not affected by AQP1 deletion. These data established a dietary fat misprocess-ing defect in young AQP1-null mice whose etiology is not fully resolved, although bile secretion is not impaired in adult AQP1-deficient mice. Also, direct measurements of water permeability and fluid secretion in cultures of mouse cholangiocytes do not support a significant role of AQP1 in bile secretion.173

Several studies reported AQP1 expression in pancreatic ducts and/or pancreatic microvasculature;174–178 however, the unimpaired pancreatic fluid secretion in AQP1-null mice raises doubt about the physiological significance of pancreatic AQP1 expression, at least in mice. AQP5 and AQP8 expression in pancreas were also found in several of these descriptive expression studies, although direct func-tional studies in knockout mice have not been done.


As done for AQP1-null mice, the AQP8-null mice were stressed by a high-fat diet to expose potentially subtle defects in hepatobiliary function. Weight gain in wild-type and AQP8-null mice on a high-fat diet was similar, and the AQP8-null mice did not develop steatorrhea or abnormali-ties in serum lipid profile, liver function tests, or pancreatic enzymes. It was concluded that AQP8 does not have an essen-tial role in hepato/biliary/pancreatic function, although a more definitive conclusion will require direct assessment of the secretion rate and composition of bile and pancreatic fluid. The only significant difference between wild-type and AQP8-null mice on a high-fat diet was mildly elevated plasma triglyceride and cholesterol concentrations in the AQP8-null mice, whose etiology and significance are unclear. In a recent study, AQP8 knockdown in HepG2 cell cultures was associated with impairment in canalicular water handling,179 although it is difficult to extrapolate results from a cell culture model to in vivo physiology. Thus, a physiologically signifi-cant role of AQP1 and AQP8 in hepatobiliary and pancreatic secretory function remains unproven.

There is recent evidence for possible involvement of AQP9 in metabolism-related hepatocyte functions. AQP9 appears to be a relatively non-selective AQP that transports water, glycerol, and a variety of other small polar solutes. Knockout mice lacking AQP9 manifest a defect in hepatic glycerol update, with increased plasma glycerol and tri-glycerides in AQP9-null mice, and abnormal blood glu-cose regulation in a strain of obese mice into which was introduced the AQP9 gene deletion.180 A possible role for AQP9 in glucose metabolism was speculated, although no follow-up work has been reported. An interesting alter-native role for AQP9 in hepatocytes has been suggested based on the observation of reduced arsenic clearance and increased arsenic toxicity in AQP9 knockout mice.181 Mice given sodium arsenite showed arsenic accumulation in liver with reduced fecal and urinary excretion, suggesting that hepatocyte AQP9 is involved in arsenic exit from liver. However, it remains unclear whether the observed phe-notype is a direct consequence of AQP9 gene deletion in hepatocytes or a secondary phenomenon related to altera-tions in other hepatocyte genes involved in arsenic detoxi-fication in AQP9 deficiency.

65.4.4 Gallbladder

The gallbladder epithelium is perhaps the most intensely investigated area of the GI tract in relation to water trans-port, in part because it is relatively easy to study, because it consists of a simple, relatively flat epithelium with large cuboidal cells that are homogenous in size and functional properties. Also, the gallbladder is a blind sac allowing assessment of water transport by relatively simple gravi-metric techniques. Studies on rabbit gallbladder formed the original experimental basis for the standing gradient

model of fluid absorption in leaky epithelia.7,182–189 Follow-up studies in mammalian and Necturus gallblad-der investigated the osmotic water permeability and mor-phology of the apical (lumen-facing) membrane and the epithelium as a whole in an effort to measure the primary parameters to differentiate among the various fluid trans-port models for transepithelial fluid transport.190–200

The gallbladder is primarily an absorptive epithelium that functions to concentrate sodium salts of bile acids by near-isotonic fluid absorption from the gallbladder lumen. Transcellular water flux across highly water-permeable api-cal and basolateral cell membranes is the prevailing view on the mechanism of fluid absorption across the gallblad-der epithelium.192 However, this view has been questioned based on both methodological and theoretical considera-tions (see Section 65.2). AQP1 is prominently expressed on the apical plasma membrane of gallbladder epithe-lial cells.106,201 We recently investigated a possible role for AQP1 in mouse gallbladder using knockout mice.106 Transepithelial osmotic water permeability (Pf) was meas-ured in freshly isolated gallbladder sacs from the kinetics of luminal calcein self-quenching in response to an osmotic gradient. Pf was very high (0.12 cm/s) in gallbladders from wild-type mice and independent of osmotic gradient size and direction. Although gallbladders from AQP1 knockout mice had similar size and morphology to those from wild-type mice, their Pf was reduced by ~10-fold. Apical plasma membrane water permeability was greatly reduced in AQP1-deficient gallbladders, as measured by cytoplasmic calcein quenching in perfluorocarbon-filled, inverted gallbladder sacs. However, neither bile osmolality nor bile salt concen-tration differed in gallbladders from wild-type versus AQP1 knockout mice. The data indicated constitutively high water permeability in mouse gallbladder epithelium involving transcellular water transport through AQP1. However, the similar bile salt concentration in gallbladders from AQP1 knockout mice argued against a physiologically important role for AQP1 in mouse gallbladder.

65.4.5 Small Intestine

The normally functioning small intestine carries out sub-stantial net absorption of fluid (~6–7 L per day in humans), which represents a combination of absorptive and secre-tory fluid fluxes. In response to various stimuli and in disease states such as bacterial/viral infection, net fluid absorption switches to fluid secretion that can result in diarrhea.202 The small intestine is a geometrically com-plex epithelia with many villi protruding into the intestinal lumen, increasing the effective surface area of the epithe-lium for solute and water absorption. Interspersed between the villi are the “gland-like” crypts of Lieberkühn that form the progenitor cells for the villous tips. It has been postulated that fluid secretion occurs predominantly within


the crypt cells and fluid absorption at the villous tip. This view has begun to be revised in recent years with both absorptive and secretory functions ascribed to cells in both locations.203–216

The magnitude of fluid movement varies along the length of the small intestine with the bulk of fluid absorp-tion occurring in the jejunum and ileum. As discussed in detail in Section 65.4, the mechanism and pathway of water transport across the small intestinal epithelium remains unresolved.

Expression of at least eight AQPs in small intestine has been reported:166,217,218 AQP1 in endothelia and lacteals of small intestine and microvascular endothelia throughout the intestine; AQP3 at the basolateral membrane of the epi-thelial cells lining the villous tip of the small intestine in rat; AQP4 at the basolateral membrane of crypt epithelium; AQP5 in the apical membrane of secretory cells in duode-nal glands; AQP6 in surface epithelial cells; AQP8 at api-cal membrane of epithelia in rat small intestine; and AQP9 in goblet cells in duodenum, jejunum, ileum. Another water channel, AQP10, has been identified219,220 with tran-script expression in human duodenum and jejunum, and protein expression in the apical membrane of villus epithe-lium in ileum.221

65.4.5.1 Duodenum

A primary role of the duodenum is the protection of epi-thelium and underlying tissue from damage by gastric acid and proteases produced in the stomach. The duode-num achieves this primarily by the secretion of mucus and bicarbonate into the lumen from the epithelium and sub-mucosal glands (Brunner’s glands) forming a protective mucous-bicarbonate barrier.222–224 The mucous barrier is formed when mucins and water combine to form a bicar-bonate-rich viscoelastic gel that creates a physiochemical barrier to acid and enzymes.225

Secretion of mucous fluid from Brunner’s glands and the epithelium clearly has an important role in normal duodenal function. However, the pathway for the water component of this secretion is so far undetermined. AQP5 has been found at the apical membrane of acinar cells in Brunner’s glands226,227 suggesting a possible role in fluid secretion from glands. There is no functional data for facil-itated water transport in duodenum.

65.4.5.2 Jejunum and Ileum

Water movement in the jejunum and ileum in the healthy intestine is primarily absorptive.228 Bacterial toxins such as cholera toxin and Escherichia coli STa toxin act on enterocytes resulting in massive secretion of NaCl and a consequent reversal of net water flux from absorptive to secretory, producing diarrhea.229 The role of AQPs in fluid

absorption in the jejunum and ileum is unclear. Osmotic water permeability of the apical membrane of small intes-tinal enterocytes, when measured minimizing confounding effects of unstirred layers, is generally found to be fairly high.25,230 Early perfusion studies suggested that proxi-mal segments of small intestine have higher osmotic per-meability than distal segments.228 The proximal jejunum has been proposed to be highly water permeable to permit rapid osmotic equilibration of intestinal contents.231–233 Therefore, it has been proposed that (see Section 65.2.2) the transcellular water permeability is sufficiently high to account for the majority of water transport across the epithelium. Whether AQPs are involved in this process is unclear. One possibility is that the specialized lipid com-position of enterocyte plasma membranes and their con-voluted microvillar ultrastructure confer high transcellular water permeability in the absence of AQPs.

Another widely accepted model states that water moves across the epithelium primarily by a paracellular route across the tight junctions between neighboring cells. Since the small intestinal epithelium has low electrical resistance, low reflection coefficients for small solutes,234 and high paracellular solute flux, it has been proposed that the tight junctions are also permeable to water. Small intestinal tight junctions have thought to be cation selective235 and perme-able to large polar non-electrolytes. This led to the sug-gestion that osmotic gradients created by active transport of solutes along with the passive ion permeability through the tight junction results in “solvent drag” of water through the paracellular pathway (see also Section 65.2.4). Several experimental and theoretical studies34,61,236 based on the dynamics of various paracellular probes have supported the paracellular model for water movement, and the interpreta-tion of these data is still subject to considerable debate. An alternative pathway that has been proposed is that transport occurs via the SGLT cotransporter (see Section 65.2.5).

Experiments on AQP knockout mice have thus far found little difference in fluid absorption or secretion in the small intestine compared to wild-type mice, leaving the role of AQPs in the small intestine (and colon) an unresolved ques-tion. At least four AQPs have been reported in the epithelial cells; AQP4 in the basolateral membrane of enterocytes at the base of crypts in the ileum,166 AQP3 in the basolateral membrane of villous enterocytes,166,217 AQP8 in the apical membrane of both crypt and enterocytes,148 and AQP10 at the apical membrane of enterocytes in ileum.221 AQP4 is only expressed at the very base of crypts in undifferentiated enterocytes, so it is thought not to be responsible for fluid transport in the intestine. Also, it is unclear whether AQP3 is actually expressed in small intestinal enterocytes with equivocal immunostaining reported in tissues.166,217 AQP8 may be the main AQP expressed in small intestinal crypt cells;148,217 however, analysis of AQP8-null mice showed lit-tle phenotypic differences compared to wild-type mice.148


Osmotically-driven water secretion, isosmolar fluid absorp-tion, and cholera toxin-induced active fluid secretion were compared in wild-type versus AQP8-null mice. Osmotically driven water secretion was measured from luminal fluid osmolalities measured at 5 and 15 minutes after infusing a hyperosmolar solution (PBS containing 300 mM mannitol) into closed jejunal loops in vivo. Osmotic equilibration did not differ significantly in wild-type versus AQP8-null mice. Apparent transepithelial osmotic water permeability coeffi-cients, computed assuming the jejunum as a smooth cylin-der, were 0.012 and 0.014 cm/s for wild-type and AQP8-null mice, respectively. Isosmolar fluid absorption was measured from the increase in concentration of a volume marker (blue dextran) at 20 minutes after infusion of jejunal and descend-ing colonic loops with an isosmolar saline solution and showed comparable rates of isosmolar fluid absorption in wild-type versus AQP8-null mice. Computed fluid absorp-tion rates in jejunum were 2.0 0.4 and 1.7 0.4 µl/min/cm2 luminal surface for wild-type and AQP8-null mice, respectively. Cholera toxin-induced fluid secretion was also measured and no significant difference between wild-type and AQP8-null mice was found. Thus, the experimental data suggest that AQPs do not constitute a major pathway for water movement across the small intestinal epithelium.

65.4.6 Colon

Water transport in the colon is primarily absorptive and occurs against a significant osmotic and hydraulic resist-ance imposed by the luminal contents. The primary func-tions of the colon are the salvage of the remaining fluid and electrolytes entering from the small intestine, and the dehy-dration and storage of feces. The colon is a tight epithelium with substantially higher electrical resistance than the small intestine, and a much lower paracellular permeability. The colonic epithelium consists of a flat surface epithelium and numerous deep crypts containing absorptive and secretory columnar epithelial cells. Additionally, colonic structure and function vary along its length with fecal dehydration becom-ing more important distally. Early studies showed that in contrast to the small intestine the colon is able to transport fluid hypertonically, with absorbate osmolalities of 500–1000 mOsm.233 As in the small intestine the route of water movement in the colon has been the subject of ongoing debate. At least three AQPs are expressed in the colon — AQP3 and AQP4 in surface epithelial cells and AQP8 in crypt epithelial cells. There are reports of AQP8 upregula-tion in human colon in ulcerative colitis237 and downregula-tion in a mouse model allergic diarrhea,238 although there is no evidence for involvement of AQP8 in the pathophysiol-ogy of these processes.

Studies in AQP knockout mice have not supported a role for AQPs in colonic fluid absorption. AQP4 is local-ized at the basolateral membrane of surface colonic

epithelial cells with strongest expression in proximal colon. Analysis of stool water content indicated no dif-ferences in cecal stool from wild-type versus AQP4 knockout mice, although there was a small but signifi-cantly greater water content in defecated stool from the knockout mice.239 The transepithelial osmotic water per-meability coefficient (Pf) of the in vivo perfused colon was measured using 14C-polyethylene glycol as a volume marker. Pf of wild-type mice was 0.016 0.002 cm/s, independent of osmotic gradient magnitude and direc-tion, as well as the solute used to induce osmosis. Pf was significantly reduced in the AQP4 knockout mice in the proximal but not distal colon. However, despite the differences in water permeability with AQP4 dele-tion, theophylline-induced secretion was not impaired (50 9 vs. 51 8 µL/min/g). Colonic fluid absorption and secretion were also measured in AQP8 knockout mice.148 Isosmolar fluid absorption measured in closed loops of descending colon was similar in wild-type ver-sus AQP8-null mice (1.8 0.1 vs. 2.0 0.4 µL/min/cm2, respectively). Also, stool water content did not differ sig-nificantly (60 3% vs. 59 1%).

The results from AQP knockout mice suggest that AQPs play at most a minor role in water absorption in the colon, suggesting that other mechanisms are involved. A significant proportion of water absorption occurs at the surface epithelium of both proximal and distal colon,212 although the exact route of water flux is unclear. The proc-ess of fecal dehydration occurs against substantial osmotic and hydraulic resistance imposed by feces as their water content is reduced from 95 to 70%. The mechanism by which the colon carries out fecal dehydration is not yet resolved. Several studies have suggested that colonic crypts are the major site of both fluid absorption and ago-nist-stimulated fluid secretion.206,215,240 Studies by Naftalin et al.212 using agarose gel cylinders to mimic the hydrau-lic resistances imposed by feces, as well as optical stud-ies using fluorescent dextrans, indicated that colonic crypts play an important role in colonic fluid absorption. These studies led to a model of fecal dehydration, based on the original Curran and Macintosh model, in which solute absorption by colonic crypts produces a hypertonic peric-ryptal compartment outside of the crypt lumen (Figure 65.5). Distal colonic crypts are surrounded by a fenestrated membrane or “sheath” consisting of myofibroblast cells and extracellular matrix components, creating a diffusion barrier to sodium and providing mechanical rigidity to resist collapse of the crypt under negative pressure.207,211 Experiments using a fluorescent sodium indicator showed high sodium concentrations in the pericryptal space.208 The resultant osmotic gradient is proposed to drive water trans-port from the crypt lumen across a relatively water-imper-meable barrier, allowing the mouth of the crypt to act as a suction device to absorb water out of fecal material within


the colon lumen. Initial evidence for this mechanism was the demonstration by photobleaching experiments of con-vective fluid flow within crypts.209 Additional evidence for a role for the “pericryptal sheath” in modulating fluid absorption comes from studies showing that pro-absorp-tive stimuli, such as high plasma rennin-angiotensin II-aldosterone, correlate with a trophic effect on the peric-ryptal sheath and increased pericryptal sodium concen-trations.241 However, although the suction hypothesis is currently the only mechanism proposed to account for fecal dehydration, direct measurements of crypt suction and water permeability are needed to examine this interest-ing mechanism further.

Motivated by the role of AQP3 in cell proliferation in skin and cornea, as described earlier, we investigated the involvement of AQP3 in enterocyte proliferation using a mouse model of inflammatory bowel disease.127 AQP3 is expressed at the basolateral plasma membrane of colono-cytes. AQP3-null mice given dextran sulfate developed severe colitis after 3 days, with colonic hemorrhage, marked epithelial cell loss, and death. Wild-type mice, which had comparable initial colonic damage as assessed by cell

apoptosis, developed remarkably less severe colitis, sur-viving 8 days. Cell proliferation was greatly reduced in AQP3-null mice as shown by BrdU incorporation. Oral glycerol administration significantly improved survival and reduced the severity of colitis in AQP3-null mice, suggest-ing a role for AQP3-faciliated glycerol transport as found in epidermis. Remarkable differences were also seen in an intracolonic acetic acid model of colitis. These results impli-cated a role for AQP3 in enterocyte proliferation, suggesting AQP3 as a potential target for therapy of intestinal diseases associated with rapid enterocyte turnover.

65.4.7 Gastrointestinal Tumors

As previously discussed, AQPs are involved in the migra-tion, spread, and proliferation of some tumor types. Several studies reported expression of AQPs in human GI tumors, such as AQP4 in gastric epithelial tumors,242 AQP5 in intes-tinal adenocarcinomas,243,244 AQP8 in colorectal tumors,245 and AQP1 in liver tumors.246 Further research is needed to investigate whether AQPs are protumorigenic in GI cancers and the possible use of AQP inhibitors, when available, for therapy of GI tumors.

65.5 SUMMARY AND PERSPECTIVE

All membranes, including those lacking AQPs, are moder-ately water permeable such that “highly” water-permeable membranes are no more than ~50-fold more water perme-able than “water-impermeable” membranes. Slow water transport occurs across plasma membranes in all cells for housekeeping functions such as volume regulation. AQPs probably have little importance in such processes. Phenotype studies in AQP knockout mice suggest that AQPs can have physiological importance for rapid water transport in response to osmotic gradients created by continuous flow (as in kidney) or active salt pumping (as in saliva secretion). AQPs are also thought to be involved in electrically excit-able tissues, perhaps by dissipating osmotic gradients cre-ated during rapid potassium recycling. The involvement of AQPs in cell migration appears to be significant not only in angiogenesis, but in a wide range of processes includ-ing tumor spread, glial scarring, wound repair and immune cell chemotaxis. The aquaglyceroporins (AQP3 and AQP7), which transport glycerol as well as water, appear to have physiological importance in mechanisms that require high cell membrane glycerol permeability such as cell metabo-lism/proliferation, epidermal hydration, and adipocyte trig-lyceride biosynthesis. However, phenotype studies revealed that the functioning of many tissues expressing AQPs, such as lung, airways, various microvascular beds, and skeletal muscle, are not impaired by AQP deletion. The reasons for the expression of AQPs in many tissues without apparent functional significance remain unclear.

Pericryptalsheath

Myofibroblastcells

Colon crypt

High [Na+]

Suctiontension

Fluid flow

Surface

Colon lumen

Na+

H2O

Epithelium

FIGURE 65.5 Model of colonic crypt fluid absorption. Na is transported across the crypt epithelium in to the surrounding interstitium. The pericryptal sheath, consisting of myofibroblast cells and extracellular matrix elements, provides a barrier to Na movement creating a hyper-tonic compartment. Water is transported across the crypt creating a suc-tion tension at the crypt opening. This tension causes fluid influx from the lumen allowing dehydration of the luminal fecal contents.


There remain many questions to be addressed about water transporting mechanisms in GI physiology. Although fluid secretion by GI organs is now reasonably well under-stood, some basic mechanisms of how fluid is absorbed in small intestine and colon remain unresolved. The contro-versial role of sodium-coupled solute transporters in fluid absorption in small intestine needs to be resolved, as does the crypt suction mechanism for fecal dehydration in colon. The finding of numerous AQPs in multiple organs of the GI tract without apparent function remains perplexing. Whereas AQP deletion has produced many interesting phenotypes in the kidney, CNS, eye, skin, and fat, few phenotypes have been found in GI organs. The role of AQP9 in liver func-tion requires further investigation, as does the expression of other possibly novel water-transporting proteins. Finally, possible roles of AQPs in diseases of the GI tract and the development of novel therapies based on AQP function war-rant evaluation. For example, is AQP expression altered in biliary and pancreatic disease, and in various diarrheas, and can pharmacological modulation of AQP expression/function by novel agents or gene delivery alter the course of diarrheas, inflammatory bowel disease, hepatic fibrosis, Sjögren's syndrome, and other GI disorders? The investiga-tion of molecular water-transporting mechanisms in the GI tract has been an understudied area with potential for sig-nificant advances.

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Physiology of the Gastrointestinal Tract || Water Transport in the Gastrointestinal Tract

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