Standing-gradient osmotic flow. A mechanism for coupling of water and solute transport in epithelia

At the ultrastructural level, epithelia performing solute-linked water transport possess long, narrow channels open at one end and closed at the other, which may constitute the fluid transport route (e.g., lateral intercellular spaces, basal infoldings, intracellular canaliculi, and brush-border microvilli). Active solute transport into such folded structures would establish standing osmotic gradients, causing a progressive approach to osmotic equilibrium along the channel's length. The behavior of a simple standing-gradient flow system has therefore been analyzed mathematically because of its potential physiological significance. The osmolarity of the fluid emerging from the channel's open end depends upon five parameters: channel length, radius, and water permeability, and solute transport rate and diffusion coefficient. For ranges of values of these parameters encountered experimentally in epithelia, the emergent osmolarity is found by calculation to range from isotonic to a few times isotonic; i.e., the range encountered in epithelial absorbates and secretions. The transported fluid becomes more isotonic as channel radius or solute diffusion coefficient is decreased, or as channel length or water permeability is increased. Given appropriate parameters, a standing-gradient system can yield hypertonic fluids whose osmolarities are virtually independent of transport rate over a wide range, as in distal tubule and avian salt gland. The results suggest that water-to-solute coupling in epithelia is due to the ultrastructural geometry of the transport route.     

Functional consequences of ultrastructural geometry in "backwards" fluid-transporting epithelia

Many fluid-transporting epithelia possess dead-end, long, and narrow channels opening in the direction to which fluid is being transported (basal infoldings, lateral intercellular spaces, etc.). These channels have been thought to possess geometrical significance as standing-gradient flow systems, in which active solute transport into the channel makes the channel contents hypertonic and permits water-to-solute coupling. However, some secretory epithelia (choroid plexus, Malpighian tubule, rectal gland, etc.) have "backwards" channels opening in the direction from which fluid is being transported. It is shown that these backwards channels can function as standing-gradient flow systems in which solute transport out of the channel makes the channel contents hypotonic and results in coupled water flow into the channel mouth. The dependence of the transported osmolarity (isotonic or hypertonic) on channel radius, length, and other parameters is calculated for backwards channels for values of these parameters in the physiological range. In addition to backwards channels' being hypotonic rather than hypertonic, they are predicted to differ from "forwards" channels in that some restrictions are imposed by the problem of solute exhaustion, and in the presence of a sweeping-in effect on other solutes which limits the solutes that may be transported.     

Mechanism and Control of Fluid Secretion

Fluid secretion and reabsorption by a variety of plant and animal tissues appear to be accomplished by osmotic coupling between solute transport and water movement. The local osmosis model suggests that active accumulation of solutes within narrow folds at the cell surface may produce the local gradients that generate water flow. Both micropuncture techniques and electron-probe X-ray microanalysis have established that local osmotic gradients occur in absorptive epithelia, but they have not as yet been detected in secretory tissues.Hormonal control of secretion involves stimulation of solute pumps and adjustments of permeability to non-transported solutes. Since hormone receptors and pumps are often located on opposite surfaces of the cell, intracellular second messengers convey the secretory signal through cytoplasm. Much has been learned by study of insect tissues that are anatomically simple and that function for long periods in vitro. Aspects of hormone-receptor interaction have been explored, including the action of halluninogenic molecules. In insect salivary glands cyclic AMP appears to stimulate cation transport, while calcium increases anion permeability. The various second messengers probably interact with each other in complex feedback loops that stabilize the system and make it quickly responsive to hormone. Cyclic AMP may stimulate release of calcium from mitochondria. Unresolved is the way second messengers alter properties of the cell surface.     

Epithelial water transport in a balanced gradient system.

The relationship between epithelial fluid transport, standing osmotic gradients, and standing hydrostatic pressure gradients has been investigated using a perturbation expansion of the governing equations. The assumptions used in the expansion are: (a) the volume of lateral intercellular space per unit volume of epithelium is small; (b) the membrane osmotic permeability is much larger than the solute permeability. We find that the rate of fluid reabsorption is set by the rate of active solute transport across lateral membranes. The fluid that crosses the lateral membranes and enters the intercellular cleft is driven longitudinally by small gradients in hydrostatic pressure. The small hydrostatic pressure in the intercellular space is capable of causing significant transmembrane fluid movement, however, the transmembrane effect is countered by the presence of a small standing osmotic gradient. Longitudinal hydrostatic and osmotic gradients balance such that their combined effect on transmembrane fluid flow is zero, whereas longitudinal flow is driven by the hydrostatic gradient. Because of this balance, standing gradients within intercellular clefts are effectively uncoupled from the rate of fluid reabsorption, which is driven by small, localized osmotic gradients within the cells. Water enters the cells across apical membranes and leaves across the lateral intercellular membranes. Fluid that enters the intercellular clefts can, in principle, exit either the basal end or be secreted from the apical end through tight junctions. Fluid flow through tight junctions is shown to depend on a dimensionless parameter, which scales the resistance to solute flow of the entire cleft relative to that of the junction. Estimates of the value of this parameter suggest that an electrically leaky epithelium may be effectively a tight epithelium in regard to fluid flow.     

Osmotic water flow in leaky epithelia

I review three currently unsolved and controversial problems in understanding solute-linked water transport in epithelia. 1. Values of osmotic water permeability (Posm) calculated from steady-state osmotic flow in response to a gradient of a probe molecule tend to be underestimates, because of three unstirred-layer (USL) effects. These are: dissipation of the probe's gradient by diffusion in USL's; reduction of the probe's gradient, due to the sweeping-away effect of water flow generated by the probe itself; and solute polarization (creation of an opposing gradient of an initially symmetrically distributed solute by the sweeping-away effect). These errors increase with probe permeability, USL thickness, Posm, and concentration ratio of symmetrically distributed solute to probe, and vary inversely as the fractional area available for water flow (e.g., lateral intercellular space width). The form of an osmotic transient, and the possibility of extracting a true Posm value from the transient, depend on the relative values of three time constants: those for solute diffusion in USL's, for solute polarization by water flow in USL's and for measuring water flow. Sweeping-away effects cause major underestimates (by one or more orders of magnitude) in epithelial Posm determinations, as shown by apparent streaming potentials during osmotic flow and by transiently reversed flows after removal of the proble. True Posm values for leaky epithelia probably exceed 10(-3) or 10(-2) cm/sec.osm. The necessary conditions for resolving osmotic transients are set out. 2. I illustrate the difficulties in deciding what fraction of transepithelial water flow is via the cells, and what fraction via the junctions. There is no existing method for answering this question. 3. Controversies about the validity, or need for modification, of the standing-gradient theory are discussed. Progress in this field requires new methods: to resolve osmotic transients; to separate transcellular and transjunctional water flows; and to measure solute concentrations in lateral intercellular spaces directly.     

Coupled water transport in standing gradient models of the lateral intercellular space.

A standing gradient model of the lateral intercellular space is presented which includes a basement membrane of finite solute permeability. The solution to the model equations is estimated analytically using the "isotonic convection approximation" of Segel. In the case of solute pumps uniformly distributed along the length of the channel, the achievement of isotonic transport depends only on the water permeability of the cell membranes. The ability of the model to transport water against an adverse osmotic gradient is the sum of two terms: The first term is simply that for a well-stirred compartment model and reflects basement membrane solute permeability. The second term measures the added strength due to diffusion limitation within the interspace. It is observed, however, that the ability for uphill water transport due to diffusion limitation is diminished by high cell membrane water permeability. For physiologically relevant parameters, it appears that the high water permeability required for isotonic transport renders the contribution of the standing gradient relatively ineffective in transport against an osmotic gradient. Finally, when the model transports both isotonically and against a gradient, it is shown that substantial intraepithelial solute polarization effects are unavoidable. Thus, the measured epithelial water permeability will grossly underestimate the water permeability of the cell membranes. The accuracy of the analytic approximation is demonstrated by numerical solution of the complete model equations.     

PATHS OF TRANSTUBULAR WATER FLOW IN ISOLATED RENAL COLLECTING TUBULES

The cells of perfused rabbit collecting tubules swell and the intercellular spaces widen during osmotic flow of water from lumen to bath induced by antidiuretic hormone (ADH). Ouabain had no influence on these changes. In the absence of net water flow intercellular width was unaffected when tubules were swollen in hypotonic external media. Therefore, during ADH-induced flow widening of intercellular spaces is not a consequence of osmotic swelling of a closed intercellular compartment containing trapped solutes, but rather is due to flow of solution through the channel. Direct evidence of intercellular flow was obtained. Nonperfused tubules swollen in hypotonic media were reimmersed in isotonic solution with resultant entry of water into intercellular spaces. The widened spaces gradually collapsed completely. Spaces enlarged in this manner could be emptied more rapidly by increasing the transtubular hydrostatic pressure difference. In electron micrographs a path of exit of sufficient width to accommodate the observed rate of fluid flow was seen at the base of the intercellular channel. It is concluded that the intercellular spaces communicate with the external extracellular fluid and that water, having entered the cells across the luminal plasma membrane in response in ADH, leaves the cells by osmosis across both the lateral and basilar surface membranes.     

ULTRASTRUCTURAL STUDIES OF VASOPRESSIN EFFECT ON ISOLATED PERFUSED RENAL COLLECTING TUBULES OF THE RABBIT

Isolated cortical collecting tubules from rabbit kidney were studied during perfusion with solutions made either isotonic or hypotonic to the external bathing medium. Examination of living tubules revealed a reversible increase in thickness of the cellular layer, prominence of lateral cell membranes, and formation of intracellular vacuoles during periods of vasopressin-induced osmotic water transport. Examination in the electron microscope revealed that vasopressin induced no changes in cell structure in collecting tubules in the absence of an osmotic difference and significant bulk water flow across the tubule wall. In contrast, tubules fixed during vasopressin-induced periods of high osmotic water transport showed prominent dilatation of lateral intercellular spaces, bulging of apical cell membranes into the tubular lumen, and formation of intracellular vacuoles. It is concluded that the ultrastructural changes are secondary to transepithelial bulk water flow and not to a direct effect of vasopressin on the cells, and that vasopressin induces osmotic flow by increasing water permeability of the luminal cell membrane. The lateral intercellular spaces may be part of the pathway for osmotically induced transepithelial bulk water flow.     

FLUID TRANSPORT IN THE RABBIT GALLBLADDER : A Combined Physiological and Electron Microscopic Study

The fine structure of the rabbit gallbladder has been studied in specimens whose functional state was undetermined, which were fixed either in situ or directly after removal from the animal; in specimens whose rate of fluid absorption was determined, either in vivo or in vitro, immediately prior to fixation; and in specimens from bladders whose absorptive function was experimentally altered in vitro. Considerable variation was found in the width of the epithelial intercellular spaces in the bladders whose functional state was undefined. In bladders known to be transporting fluid, either in vivo or in vitro, the intercellular spaces were always distended, as were the subepithelial capillaries. This distension was greatest in bladders which had been functioning in vitro. When either Na⁺ or Cl^- was omitted from the bathing media, there was no fluid transport across the wall of the gallbladder studied in vitro. The epithelial intercellular spaces of biopsies taken from several bladders under these conditions were of approximately 200 A width except for minor distension at the crests of mucosal folds. The addition of the missing ion rapidly led to the reestablishment of fluid transport and the distension of the intercellular spaces throughout most of the epithelium of these bladders. Studies of sodium localization (by fixation with a pyroantimonate-OsO₄ mixture) showed high concentrations of this ion in the distended intercellular spaces. Histochemical studies of ATPase activity showed that this enzyme was localized along the lateral plasma membrane of the epithelial cells. The analogy is drawn between the structure of the gallbladder mucosa and a serial membrane model proposed by Curran to account for coupled solute-solvent transport across epithelia. It is concluded that the intercellular compartment fulfills the conditions for the middle compartment of the Curran model and that active transport of solute across the lateral plasma membrane into the intercellular space may be responsible for fluid absorption by the gall bladder.     

Models for coupling of salt and water transport; Proximal tubular reabsorption in Necturus kidney

Models for coupling of salt and water transport are developed with two important assumptions appropriate for leaky epithelia. (a) The tight junction is permeable to both sale and water. (b) Active Na transport into the lateral speces is assumed to occur uniformly along the length of the channel. The proposed models deal specifically with the intraepithelial mechanism of proximal tubular resbsorption in the Necturus kidney although they have implications for epithelial transport in the gallbladder and small intestine as well. The first model (continuous version) is similar to the standing gradient model devised by Diamond and Bossert but used different boundary conditions. In contrast to Diamond and Bossert's model, the predicted concentration profiles are relatively flat with no sizable gradients along the interspace. The second model (compartment version) expands Curran's model of epithelial salt and water transport by including additional compartments and considering both electrical and chemical driving forces for individual Na and Cl ions as well as hydraulic and osmotic driving forces for water. In both models, ion and water fluxes are investigated as a function of the transport parameters. The behavior of the models is consistent with previously suggested mechanisms for the control of net transport, particularly during saline diuresis. Under all conditions the predicted ratio of net solute to solvent flux, or emergent concentration, deviates from exact isotonicity (except when the basement membrane has an appreciable salt reflection coefficient). However, the degree of hypertonicity may be small enough to be experimentally indistinguishable from isotonic transport.     

Apparent Transport of Water by Insect Excretory Systems

Insects are capable of producing strongly hyperosmotic urinebut most species do not possess the anatomical equivalent ofthe mammalian kidney's couiitercurrent system. Concentrationof the excreta occurs in the rectum where water is absorbedagainst increasing osmotic gradients without strict dependenceon simultaneous absorption of solute. Properties of this processare reviewed. It is currently postulated that this apparenttransport of water is driven by local transport and recyclingof solute within the lateral intercellular spaces of the epitheliumof the rectal pad. The most concentrated excreta so far reported are those of themealworm, Tenebrio molitor. This species possesses a cryptonephridialcomplex in which the posterior end of the malpighian tubulesis closely applied to the rectum and both are enclosed withina complex membranous sheath. Active transport of potassium chlorideby the malpighian tubules into the complex creates a local highosmotic pressure within the complex which is responsible, inpart if not completely, for removal of water from the rectallumen. This system bears some resemblance to the countercurrentsystem of the mammalian kidney.     

The effect of osmotically induced water flows on the permeability and ultrastructure of the rabbit gallbladder

Summary The purpose of these experiments was to determine the effect of osmotic gradients on the permeability of the rabbit gallbladder. Increasing the tonicity of the mucosal solution reduced the permeability of the gallbladder to both ions and nonelectrolytes, whereas there was no significant effect when the serosal solution was made hypertonic. These results cannot be explained by solvent/solute interactions in either the epithelial membranes or the unstirred layers. Associated with the changes in permeability was the appearance of the transport number effect and current induced resistance changes. Morphological studies of the gallbladder under these conditions showed that the extracellular spaces of the epithelium and the rest of the wall dilate in the presence of osmotic flow to the serosa, but that the spaces collapse when the flow is in the opposite direction. Reconstruction of the permeability changes from the dimensions of the tissue show that all the physiological phenomena are accounted for by the changes in morphology, the dominant effect being in the lateral intercellular spaces. These results suggest that the lateral spaces are a common pathway shared by all solutes crossing the epithelium, and that diffusion along these spaces becomes rate limiting as the spaces collapse.     

The role of the lateral intercellular spaces and solute polarization effects in the passive flow of water across the rabbit gallbladder

Summary Osmotic water flows were measured acrossin vitro preparations of the rabbit gallbladder by a gravimetric technique. The bladders exhibited asymmetrical osmotic behavior, in which theL _p (hydraulic conductivity) for water flow from mucosa to serosa was up to four times greater than theL _p for water flow in the opposite direction. This result is similar to the effects of osmotic gradients on ion and nonelectrolyte permeability reported in the first paper. As in the case of solute permeability, these changes inL _p are accounted for by changes in the dimensions of the lateral intercellular spaces of the epithelium. These spaces are thus a final common pathway for the movement of both solutes and water across the epithelium. We also observed osmotic flow transients in which the initialL _p was about an order of magnitude greater than the steady stateL _p . These transients are largely explained by solute polarization in the unstirred layers adjacent to the epithelial membranes. A comparison between streaming potentials and water flows showed that streaming potentials are directly proportional to the rate of flow only over a limited range. These observations are readily explained on the basis of structural changes and solute polarization effects. Finally, the routes of water flow across epithelia are discussed in the light of our observations.     

On the influence of a leaky tight junction on water and solute transport in epithelia

The role of a leaky tight junction in epithelia is examined by considering the flow of water and solute through a channel consisting of two sections representing the intercellular space and tight junction. Two cases are considered, flow through a channel with a circular cross-section and flow between parallel planes. Analytical solutions are obtained using the isotonic convection approximation. The flow is driven by active transport of solute and imposed concentration and pressure differences. Particular attention is paid to the flux of solute through the tight junction. It is shown that the shape of the channel cross-section is important.The theory is applied to the rat proximal tube epithelium. It is deduced that the emergent osmolarity is close to that predicted for a closed tight junction, but that transepithelial hydrostatic pressure differences are potentially important. The influence of transepithelial concentration differences appears to be unimportant in this model.     

Determinants of epithelial cell volume

Epithelial cell volume is determined by the concentration of intracellular, osmotically active solutes. The high water permeability of the cell membrane of most epithelia prevents the establishment of large osmotic gradients between the cell and the bathing solutions. Steady-state cell volume is determined by the relative rates of solute entry and exit across the cell membranes. Inhibition of solute exit leads to cell swelling because solute entry continues; inhibition of solute entry leads to cell shrinkage because solute exit continues. Cell volume is then a measure of the rate and direction of net solute movements. Epithelial cells are also capable of regulation of the rate of solute entry and exit to maintain intracellular composition. Feedback control of NaCl entry into Necturus gallbladder epithelial cells is demonstrable after inhibition of the Na,K-ATPase or reduction in the NaCl concentration of the serosal bath. Necturus gallbladder cells respond to a change in the osmolality of the perfusion solution by rapidly regulating their volume to control values. This regulatory behavior depends on the transient activation of quiescent transport systems. These transport systems are responsible for the rapid readjustments of cell volume that follow osmotic perturbation. These powerful transporters may also play a role in steady-state volume regulation as well as in the control of cell pH.     

Transport of Salt and Water in Rabbit and Guinea Pig Gall Bladder

A simple and reproducible method has been developed for following fluid transport by an in vitro preparation of mammalian gall bladder, based upon weighing the organ at 5 minute intervals. Both guinea pig and rabbit gall bladders transport NaCl and water in isotonic proportions from lumen to serosa. In the rabbit bicarbonate stimulates transport, but there is no need for exogenous glucose. The transport rate is not affected by removal of potassium from the bathing solutions. Albumin causes a transient weight loss from the gall bladder wall, apparently by making the serosal smooth muscle fibers contract. Active NaCl transport can carry water against osmotic gradients of up to two atmospheres. Under passive conditions water may also move against its activity gradient in the presence of a permeating solute. The significance of water movement against osmotic gradients during active solute transport is discussed.     

The Mechanism of Isotonic Water Transport

The mechanism by which active solute transport causes water transport in isotonic proportions across epithelial membranes has been investigated. The principle of the experiments was to measure the osmolarity of the transported fluid when the osmolarity of the bathing solution was varied over an eightfold range by varying the NaCl concentration or by adding impermeant non-electrolytes. An in vitro preparation of rabbit gall bladder was suspended in moist oxygen without an outer bathing solution, and the pure transported fluid was collected as it dripped off the serosal surface. Under all conditions the transported fluid was found to approximate an NaCl solution isotonic to whatever bathing solution used. This finding means that the mechanism of isotonic water transport in the gall bladder is neither the double membrane effect nor co-diffusion but rather local osmosis. In other words, active NaCl transport maintains a locally high concentration of solute in some restricted space in the vicinity of the cell membrane, and water follows NaCl in response to this local osmotic gradient. An equation has been derived enabling one to calculate whether the passive water permeability of an organ is high enough to account for complete osmotic equilibration of actively transported solute. By application of this equation, water transport associated with active NaCl transport in the gall bladder cannot go through the channels for water flow under passive conditions, since these channels are grossly too impermeable. Furthermore, solute-linked water transport fails to produce the streaming potentials expected for water flow through these passive channels. Hence solute-linked water transport does not occur in the passive channels but instead involves special structures in the cell membrane, which remain to be identified.     

Optimizing automated characterization of liver fibrosis histological images by investigating color spaces at different resolutions

Background

7D-cadherins like LI-cadherin are cell adhesion molecules and represent exceptional members of the cadherin superfamily. Although LI-cadherin was shown to act as a functional Ca²⁺-dependent adhesion molecule, linking neighboring cells together, and to be dysregulated in a variety of diseases, the physiological role is still enigmatic. Interestingly 7D-cadherins occur only in the lateral plasma membranes of cells from epithelia of water transporting tissues like the gut, the liver or the kidney. Furthermore LI-cadherin was shown to exhibit a highly cooperative Ca²⁺-dependency of the binding activity. Thus it is tempting to assume that LI-cadherin regulates the water transport through the epithelium in a passive fashion by changing its binding activity in dependence on the extracellular Ca²⁺.

Results

We developed a simple mathematical model describing the epithelial lining of a lumen with a content of variable osmolarity covering an interstitium of constant osmolarity. The width of the lateral intercellular cleft was found to influence the water transport significantly. In the case of hypertonic luminal content a narrow cleft is necessary to further increase concentration of the luminal content. If the cleft is too wide, the water flux will change direction and water is transported into the lumen. Electron microscopic images show that in fact areas of the gut can be found where the lateral intercellular cleft is narrow throughout the lateral cell border whereas in other areas the lateral intercellular cleft is widened.

Conclusions

Our simple model clearly predicts that changes of the width of the lateral intercellular cleft can regulate the direction and efficiency of water transport through a simple epithelium. In a narrow cleft the cells can increase the concentration of osmotic active substances easily by active transport whereas if the cleft is wide, friction is reduced but the cells can hardly build up high osmotic gradients. It is now tempting to speculate that 7D-cadherins, owing to their location and their Ca²⁺-dependence, will adapt their binding activity and thereby the width of the lateral intercellular cleft automatically as the Ca²⁺-concentration is coupled to the overall electrolyte concentration in the lateral intercellular cleft. This could provide a way to regulate the water resorption in a passive manner adapting to different osmotic conditions.     

Correlation of structural changes at different levels of the jejunal villus with positive net water transport in vivo and in vitro.

Experiments were done for indentification and localization of certain structural changes at different levels of jejunal villus of the hamster during positive and negative water transport across the intestine in vivo and in vitro. Positive transport occurred when the mucosal surface of the intestine was bathed (in vitro experiments) or perfused (in vivo experiments) with isotonic Krebs-Ringer bicarbonate solution containing 10 mM glucose, and negative water transport was achieved by rendering this solution hypertonic with 150 mM mannitol. Results indicate that during positive net water transport the intestine in vivo transported more fluid and exhibited a more conspicuous dilatation of the lateral intercellular spaces (L.I.S.) than did the in vitro preparation. Dilatation of the L.I.S. in both preparations was present only in the apical part of the villus, suggesting that this is the principal site of water absorption. When the mucosal solution was made hypertonic with mannitol, the L.I.S. in the in vivo intestine totally collapsed, whereas in the in vitro intestine these spaces remained open very slightly. These morphological changes correspond well with our finding that in the presence of the hypertonic mucosal solution there was a greater net negative water transport in vivo than in vitro. Incubation of the intestine in the isotonic mucosal solution produced subnuclear swelling of the mid-villus epithelial cells, and this morphological change was associated with an increase in the water content of the tissue. Perfusion of the in vivo intestine with the isotonic solution produced neither the swellings nor the increase in water content of the tissue. In the presence of hypertonic mucosal solution there was a water loss from the tissue both in vivo and in vitro, and these swellings were not observed. These results are discussed in relation to intestinal sugar transport and to the maturity of the epithelial cells, and it is concluded that transport studies on in vitro preparations may provide valid information on a qualitative basis, if not on a strictly quantitative basis.     

Corneal endothelium transports fluid in the absence of net solute transport

The corneal endothelium transports fluid from the corneal stroma to the aqueous humor, thus maintaining stromal transparency by keeping it relatively dehydrated. This fluid transport mechanism is thought to be driven by the transcellular transports of HCO(3)(-) and Cl(-) in the same direction, from stroma to aqueous. In parallel to these anion movements, for electroneutrality, there are paracellular Na(+) and transcellular K(+) transports in the same direction. The resulting net flow of solute might generate local osmotic gradients that drive fluid transport. However, there are reports that some 50% residual fluid transport remains in nominally HCO(3)(-) free solutions. We have examined the driving force for this residual fluid transport. We confirm that in nominally HCO(3)(-) free solutions, 48% of control fluid transport remains. When in addition Cl(-) channels are inhibited, 30% of control fluid movement still remains. Addition of a carbonic anhydrase inhibitor has no further effect. These manipulations combined inhibit the transcellular transport of all anions, without which there cannot be any net transport of solute and consequently no local osmotic gradients, yet there is residual fluid movement. Only the further addition of benzamil, an inhibitor of epithelial Na(+) channels, abolishes fluid transport completely. Our data are inconsistent with transcellular local osmosis and instead support the paradigm of paracellular fluid transport driven by electro-osmotic coupling.