Epithelial Water Transport Research Articles

Angulin-1 (LSR) Affects Paracellular Water Transport, However Only in Tight Epithelial Cells.

Water transport in epithelia occurs transcellularly (aquaporins) and paracellularly (claudin-2, claudin-15). Recently, we showed that downregulated tricellulin, a protein of the tricellular tight junction (tTJ, the site where three epithelial cells meet), increased transepithelial water flux. We now check the hypothesis that another tTJ-associated protein, angulin-1 (alias lipolysis-stimulated lipoprotein receptor, LSR) is a direct negative actuator of tTJ water permeability depending on the tightness of the epithelium. For this, a tight and an intermediate-tight epithelial cell line, MDCK C7 and HT-29/B6, were stably transfected with CRISPR/Cas9 and single-guide RNA targeting angulin-1 and morphologically and functionally characterized. Water flux induced by an osmotic gradient using 4-kDa dextran caused water flux to increase in angulin-1 KO clones in MDCK C7 cells, but not in HT-29/B6 cells. In addition, we found that water permeability in HT-29/B6 cells was not modified after either angulin-1 knockout or tricellulin knockdown, which may be related to the presence of other pathways, which reduce the impact of the tTJ pathway. In conclusion, modulation of the tTJ by knockout or knockdown of tTJ proteins affects ion and macromolecule permeability in tight and intermediate-tight epithelial cell lines, while the transepithelial water permeability was affected only in tight cell lines.

Specialized stellate cells offer a privileged route for rapid water flux in Drosophila renal tubule

Insects are highly successful, in part through an excellent ability to osmoregulate. The renal (Malpighian) tubules can secrete fluid faster on a per-cell basis than any other epithelium, but the route for these remarkable water fluxes has not been established. In Drosophila melanogaster, we show that 4 genes of the major intrinsic protein family are expressed at a very high level in the fly renal tissue: the aquaporins (AQPs) Drip and Prip and the aquaglyceroporins Eglp2 and Eglp4 As predicted from their structure, and by their transport function by expressing these proteins in Xenopus oocytes, Drip, Prip, and Eglp2 show significant and specific water permeability, whereas Eglp2 and Eglp4 show very high permeability to glycerol and urea. Knockdowns of any of these genes result in impaired hormone-induced fluid secretion. The Drosophila tubule has 2 main secretory cell types: active cation-transporting principal cells, wherein the aquaglyceroporins localize to opposite plasma membranes, and small stellate cells, the site of the chloride shunt conductance, with these AQPs localizing to opposite plasma membranes. This suggests a model in which osmotically obliged water flows through the stellate cells. Consistent with this model, fluorescently labeled dextran, an in vivo marker of membrane water permeability, is trapped in the basal infoldings of the stellate cells after kinin diuretic peptide stimulation, confirming that these cells provide the major route for transepithelial water flux. The spatial segregation of these components of epithelial water transport may help to explain the unique success of the higher insects in regulating their internal environments.

Mechanistic basis for loss of water balance in green tree frogs infected with a fungal pathogen.

Chytridiomycosis, a lethal skin disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), disrupts skin function of amphibians, interfering with ionic and osmotic regulation. To regulate fungal loads, amphibians increase their rate of skin sloughing. However, sloughing also causes a temporary loss of ionic and osmotic homeostasis due to disruption of the skin, a key osmoregulatory organ. The combined effects of increased sloughing frequency and chytridiomycosis contribute to the high rates of mortality from Bd infections. However, the mechanisms responsible for the loss of cutaneous osmotic regulation remain unknown. We measured the changes in whole animal water uptake rates, in vitro transcutaneous water fluxes across the ventral skin, and the mRNA expression of epithelial water transport proteins (aquaporins, AQPs) and junctional proteins in Bd-infected and uninfected Litoria caerulea skin. We hypothesize that infected frogs would show reduction/inhibition in cutaneous water transporters responsible for regulating water balance, and sloughing would exacerbate cutaneous water fluxes. We found that infected, nonsloughing frogs had an impaired rate of water uptake and showed increased rates of in vitro water efflux across the ventral skin. In uninfected frogs, the expression of AQPs and junction genes increased significantly with sloughing, which may assist in regulating cutaneous water movements and barrier function in the newly exposed skin. In contrast, infected frogs did not show this postsloughing increase in AQP gene expression. The combination of increased sloughing frequency, impaired water uptake rates, and increased rates of water loss likely contributes to the loss of osmotic homeostasis in frogs infected with Bd.

Lack of Vitamin D Receptor Leads to Hyperfunction of Claudin-2 in Intestinal Inflammatory Responses.

Vitamin D3 and vitamin D receptor (VDR) are involved in the pathogenesis of inflammatory bowel disease (IBD) and bacterial infection. Claudin-2 is a junction protein that mediates paracellular water transport in epithelia. Elevation of Claudin-2 is associated with active IBD. However, VDR involved in epithelial junctions under inflammation and infection remains largely unknown. We investigated the mechanisms on how VDR and Claudin-2 are related in inflamed states. Using cultured VDR-/- enteroids, human intestinal epithelial cells, VDR-/- mice with Salmonella- or DSS-colitis, and human IBD samples, we investigated the mechanisms how VDR and Claudin-2 are related in inflamed states. After Salmonella infection had taken place, we observed significantly enhanced Claudin-2 and an increased bacterial invasion and translocation. A lack of VDR regulation led to a robust increase of Claudin-2 at the mRNA and protein levels post-infection. In DSS-treated VDR-/- mice, Claudin-2 was significantly increased. Location and quantification of Claudin-2 protein in the mouse colons treated with DSS also confirmed these results. Inflammatory cytokines were significantly higher in the serum and mRNA levels in intestine, which are known to increase Claudin-2. Furthermore, in inflamed intestine of ulcerative colitis patients, VDR expression was low and Claudin-2 was enhanced. Mechanistically, we identified the enhanced Claudin-2 promoter activity through the binding sites of NF-κB and STAT in inflamed VDR-/- cells. Our studies have identified a new role for intestinal epithelial VDR in regulating barrier functions in the context of infection and inflammation.

HNF1 regulates critical processes in the human epididymis epithelium

The luminal environment of the epididymis participates in sperm maturation and impacts male fertility. It is dependent on the coordinated expression of many genes encoding proteins with a role in epithelial transport. We identified cis-regulatory elements for critical genes in epididymis function, by mapping open chromatin genome-wide in human epididymis epithelial (HEE) cells. Bioinformatic predictions of transcription factors binding to the regulatory elements suggested an important role for hepatocyte nuclear factor 1 (HNF1) in the transcriptional program of these cells. Chromatin immunoprecipitation and deep sequencing (ChIP-seq) revealed HNF1 target genes in HEE cells. In parallel, the contribution of HNF1 to the transcriptome of HEE cells was determined by RNA-seq, following siRNA-mediated depletion of both HNF1α and HNF1β transcription factors. Repression of these factors caused differential expression of 1892 transcripts (902 were downregulated and 990 upregulated) in comparison to non-targeting siRNAs. Differentially expressed genes with HNF1 ChIP-seq peaks within 20 kb were subject to gene ontology process enrichment analysis. Among the most significant processes associated with down-regulated genes were epithelial transport of water, phosphate and bicarbonate, all critical processes in epididymis epithelial function. Measurements of intracellular pH (pHi) confirmed a role for HNF1 in regulating the epididymis luminal environment.

What do aquaporin knockout studies tell us about fluid transport in epithelia?

The investigation of near-isosmotic water transport in epithelia goes back over 100 years; however, debates over mechanism and pathway remain. Aquaporin (AQP) knockouts have been used by various research groups to test the hypothesis of an osmotic mechanism as well as to explore the paracellular versus transcellular pathway debate. Nonproportional reductions in the water permeability of a water-transporting epithelial cell (e.g., a reduction of around 80-90 %) compared to the reduction in overall water transport rate in the knockout animal (e.g., a reduction of 50-60 %) are commonly found. This nonproportionality has led to controversy over whether AQP knockout studies support or contradict the osmotic mechanism. Arguments raised for and against an interpretation supporting the osmotic mechanism typically have partially specified, implicit, or incorrect assumptions. We present a simple mathematical model of the osmotic mechanism with clear assumptions and, for models based on this mechanism, establish a baseline prediction of AQP knockout studies. We allow for deviations from isotonic/isosmotic conditions and utilize dimensional analysis to reduce the number of parameters that must be considered independently. This enables a single prediction curve to be used for multiple epithelial systems. We find that a simple, transcellular-only osmotic mechanism sufficiently predicts the results of knockout studies and find criticisms of this mechanism to be overstated. We note, however, that AQP knockout studies do not give sufficient information to definitively rule out an additional paracellular pathway.

Osmoregulation and epithelial water transport: lessons from the intestine of marine teleost fish

For teleost fish living in seawater, drinking the surrounding medium is necessary to avoid dehydration. This is a key component of their osmoregulatory strategy presenting the challenge of excreting excess salts while achieving a net retention of water. The intestine has an established role in osmoregulation, and its ability to effectively absorb fluid is crucial to compensating for water losses to the hyperosmotic environment. Despite this, the potential for the teleost intestine to serve as a comparative model for detailed, integrative experimental studies on epithelial water transport has so far gone largely untapped. The following review aims to present an assessment of the teleost intestine as a fluid-transporting epithelium. Beginning with a brief overview of marine teleost osmoregulation, emphasis shifts to the processing of ingested seawater by the gastrointestinal tract and the characteristics of intestinal ion and fluid transport. Particular attention is given to acid-base transfers by the intestine, specifically bicarbonate secretion, which creates the distinctly alkaline gut fluids responsible for the formation of solid calcium carbonate precipitates. The respective contributions of these unique features to intestinal fluid absorption, alongside other recognised ion transport processes, are then subsequently considered within the wider context of the classic physiological problem of epithelial water transport.

Functional expression of the hypo‐osmosensitive channels TRPV4 and TRPM3 in mouse airway epithelium

TRPV4 and TRPM3, members of the transient receptor potential channel family, are membrane proteins that mediate Ca2+ entry in cells upon application of hypo‐osmotic solutions. This study aimed at investigating the expression and functionality of these osmosensitive channels in the epithelium of mouse intrapulmonary airways by combining confocal Ca2+ imaging and multilabel immunohistochemistry.Immunostaining revealed that TRPV4 and TRPM3 are colocalized on both ciliated cells and Clara cells, the most common cell types in the mouse airway epithelium, but also that the neuroendocrine cells, grouped as neuroepithelial bodies, are negative. Confocal live cell imaging of precision cut vibratome slices of mouse lungs loaded with Fluo‐4 showed that short term (30s) perfusion with a hypo‐osmotic solution (200 mOsm) results in a clear and reversible rise in intracellular Ca2+ in all Clara and ciliated cells.Both TRPV4 and TRPM3 channels may allow the cells to react to changes in extracellular osmolarity and therefore potentially play a central role in airway epithelial homeostasis by modulating epithelial water transport. The presented approach opens interesting new perspectives for unraveling functional aspects of diverse cell and tissue types in control lungs and disease models.Support: IWT fellowship SB/81162 (R.L); FWO grant G.0081.08 (D.A.); UA grants GOA BOF 2007 (D.A.) and KP BOF 2006 (I.B.)

Water-Transporting Proteins

Transport through lipids and aquaporins is osmotic and entirely driven by the difference in osmotic pressure. Water transport in cotransporters and uniporters is different: Water can be cotransported, energized by coupling to the substrate flux by a mechanism closely associated with protein. In the K(+)/Cl(-) and the Na(+)/K(+)/2Cl(-) cotransporters, water is entirely cotransported, while water transport in glucose uniporters and Na(+)-coupled transporters of nutrients and neurotransmitters takes place by both osmosis and cotransport. The molecular mechanism behind cotransport of water is not clear. It is associated with the substrate movements in aqueous pathways within the protein; a conventional unstirred layer mechanism can be ruled out, due to high rates of diffusion in the cytoplasm. The physiological roles of the various modes of water transport are reviewed in relation to epithelial transport. Epithelial water transport is energized by the movements of ions, but how the coupling takes place is uncertain. All epithelia can transport water uphill against an osmotic gradient, which is hard to explain by simple osmosis. Furthermore, genetic removal of aquaporins has not given support to osmosis as the exclusive mode of transport. Water cotransport can explain the coupling between ion and water transport, a major fraction of transepithelial water transport and uphill water transport. Aquaporins enhance water transport by utilizing osmotic gradients and cause the osmolarity of the transportate to approach isotonicity.

Urine concentration and avian aquaporin water channels

Although birds and mammals have evolved from primitive tetrapods and advanced divergently, both can conserve water by producing hyperosmotic urine. Unique aspects in the avian system include the presence of loopless and looped nephrons, lack of the thin ascending limb of Henle's loop, a corticomedullary osmotic gradient primarily consisting of NaCl without contribution of urea, and significant postrenal modification of final urine. The countercurrent multiplier mechanism operates between the descending and ascending limbs of Henle via recycling of a single solute (NaCl) with no water accompaniment, forming an osmotic gradient along the medullary cone. Bird kidneys and developing rat kidneys share morphological and functional characteristics. Avian kidneys express aquaporin (AQP) 1, 2, and 4 homologues that share considerable homology with mammalian counterparts, but their distribution and function may not be the same. AQP2 expression in Japanese quail (q) evolves in the collecting duct of early metanephric kidneys and continues to increase in intensity and distribution during nephrogenesis and maturation. qAQP2 mRNA and protein are increased by arginine vasotocin (avian ADH), but vasotocin-induced enhancement of cAMP production and water permeability are less marked than in mammalian kidneys. Nephrogenesis is delayed by insufficient nutrition in avian embryos and newborns and results in fewer nephrons and an impaired water balance in adults. Diabetes insipidus quail with homozygous autosomal recessive mutation and an unaffected vasotocin system have low AQP2 expression, underdeveloped medullary cones. Comparative studies will provide important insight into integrative, cellular, and molecular mechanisms of epithelial water transport and its control by humoral, neural, and hemodynamic mechanisms.

Role of cytosolic phospholipase A 2 in cytokine-stimulated prostaglandin release by human gallbladder cells

Eicosanoids are involved in gallbladder inflammation, epithelial water transport, and mucous secretion. Phospholipase A 2 enzymes liberate arachidonic acid from membrane phospholipids for the synthesis of eicosanoids. The purpose of this study was to determine the effect of selective cytoplasmic and secretory phospholipase A 2 inhibitors on basal and stimulated arachidonic acid and prostaglandin E 2 release in gallbladder cells. Western immunoblotting was employed to evaluate both cytosolic and secretory phospholipase A 2 enzymes in human gallbladder cells. Cells were incubated for 22 hours with 3H-labeled arachidonic acid. Arachidonic acid and prostaglandin E 2 release was then measured in the supernate after 2 hours of exposure to human interleukin-1β, alone or after pretreatment for 1 hour with the inhibitors. Unstimulated gallbladder cells express both 85 kDa cytosolic and 14 kDa secretory phospholipase A 2. The 85 kDa phospholipase A 2 was induced by interleukin-1β, whereas there was no apparent change in secretory phospholipase A 2 enzyme concentrations. Both the secretory phospholipase A 2 inhibitor p-bromophenylacyl bromide and the cytosolic phospholipase A 2 inhibitor arachidonyl trifluoromethyl ketone decreased basal and interleukin-1β—stimulated arachidonic acid release. In contrast, only inhibition of cytosolic phospholipase A 2 led to a decrease in interleukin-1β—stimulated prostaglandin E 2 release. Basal and interleukin-1β—stimulated arachidonic acid release appears to be the result of the activity of both cytosolic and secretory phospholipase A 2. Interleukin-1β—stimulated prostaglandin E 2 release appears to be dependent on the activity of cytosolic phospholipase A 2.

CHIP28 water channels are localized in constitutively water-permeable segments of the nephron.

The sites of water transport along the nephron are well characterized, but the molecular basis of renal water transport remains poorly understood. CHIP28 is a 28-kD integral protein which was proposed to mediate transmembrane water movement in red cells and kidney (Preston, G. M., T. P. Carroll, W. B. Guggino, and P. Agre. 1992. Science [Wash. DC]. 256:385-387). To determine whether CHIP28 could account for renal epithelial water transport, we used specific polyclonal antibodies to quantitate and localize CHIP28 at cellular and subcellular levels in rat kidney using light and electron microscopy. CHIP28 comprised 3.8% of isolated proximal tubule brush border protein. Except for the first few cells of the S1 segment, CHIP28 was immunolocalized throughout the convoluted and straight proximal tubules where it was observed in the microvilli of the apical brush border and in basolateral membranes. Very little CHIP28 was detected in endocytic vesicles or other intracellular structures in proximal tubules. Uninterrupted, heavy immunostaining of CHIP28 was also observed over both apical and basolateral membranes of descending thin limbs, including both short and long loops of Henle. These nephron sites have constitutively high osmotic water permeabilities. CHIP28 was not detected in ascending thin limbs, thick ascending limbs, or distal tubules, which are highly impermeable to water. Moreover, CHIP28 was not detected in collecting duct epithelia, where water permeability is regulated by antidiuretic hormone. These determinations of abundance and structural organization provide evidence that the CHIP28 water channel is the predominant pathway for constitutive transepithelial water transport in the proximal tubule and descending limb of Henle's loop.

Experimental Studies on the Accumulation of Subretinal Fluid1

In an attempt to develop a rabbit model for serous detachment, we damaged the pigment epithelium with low-intensity laser burns, lowered intraocular pressure and administered dibutyryl adenosine 3',5'-cyclic phosphate (cAMP), to inhibit pigment epithelial water transport. We produced only small areas of edema or very low detachment that persisted for a few days at best. These experiments suggest that it is remarkably difficult to make fluid stay in the subretinal space, and more stringent conditions will be needed to mimic the size and persistence of clinical serous detachments.

12 Water disturbances in cardiac failure

The non-osmotic stimulation of release of arginine vasopressin (AVP) seems to be the main determinant of the impaired water excretion and hyponatraemia in patients with cardiac failure. This non-osmotic stimulation of AVP release could be secondary to a decrease in stroke volume to which the ventricular receptors respond by decreasing the vagal afferent input to the hypothalamus via the mid-brain. Improvement of cardiac stroke volume would then decrease AVP release and improve water excretion. In cardiac failure, the non-osmotic stimulation of AVP release is not clearly modulated by the renin-angiotensin system or by the atrial natriuretic peptide plasma concentration. Nevertheless, physiological concentrations of atrial natriuretic peptide could inhibit the renal epithelial water transport at the collecting duct level. Water-loading and osmotic-loading experiments in patients with cardiac failure indicated that the release of AVP is still under osmotic control and favoured the concept that volume depletion in general and cardiac failure in particular may lower the osmotic threshold and increase the osmotic sensitivity to vasopressin release. Experiments using a specific vasopressin antagonist rarely indicated a vasoconstrictor role for endogenous AVP in either experimental or clinical cardiac failure. Intrarenal factors also contributed to the impaired water excretion observed in patients with cardiac failure: increased central sympathetic efferent discharge and stimulation of the renin-angiotensin-aldosterone system would be expected as a consequence of the decreased effective arterial blood volume. These effects could then decrease maximal reabsorption of solute further impairing the ability of the kidney to excrete free water. The impaired water excretion is correlated with the severity of the cardiac deterioration and thus has prognostic implications.

Osmotic reversal induces assembly of tight junction strands at the basal pole of toad bladder epithelial cells but does not reverse cell polarity.

This paper reports the effect of reversing the osmotic environment between luminal and serosal compartments of a toad urinary bladder on the polarity of assembly of tight junction strands. Toad bladders were filled with Ringer's solution (220 mOsm) and were immersed in distilled water at room temperature or at 37 degrees C. Within two minutes, new tight junction strands are assembled. The new tight junctional strands unite the basal pole of epithelial cells with the apical side of basal cells. Physiological studies show that oxytocin, a synthetic analog of antidiuretic hormone, is still capable of inducing increases in water transport in epithelia which were osmotically reversed. This capacity decreases significantly for longer periods of osmotic reversal. Osmotic reversal does not alter the original polarity of epithelial cells: the apical tight junction belt, at the apical pole, is not displaced; the freeze-fracture morphology typical of apical plasma membrane (particle-rich E faces; particle-poor P faces) is not altered; oxytocin and cyclic AMP induce aggregates which are observed only at the apical plasma membrane. Massive assembly of junctional elements occurs even in epithelia preincubated in the presence of cycloheximide (an inhibitor of protein synthesis) or of cytoskeleton perturbers. Our experiments show that the polarity of assembly of tight junction strands depends on the vectorial orientation of the osmotic environment of the epithelium.

Inhibition of vasopressin action by atrial natriuretic factor.

Atrial natriuretic factor results in diuresis in animals and humans, perhaps because atrial natriuretic factor increases renal blood flow. The possibility that this diuresis is due to direct inhibition of renal tubular epithelial water transport was examined in rabbit collecting tubules perfused in vitro. Atriopeptin III inhibition of the hydraulic conductivity response to the hormone arginine vasopressin but not to either 3'5'-cyclic adenosine monophosphate or forskolin was found. These results suggest that atriopeptin III acts proximal to cyclic adenosine monophosphate formation to directly affect vasopressin-stimulated water transport in the mammalian nephron. They also suggest a potential role for inhibition by atrial natriuretic factor of the renal response to arginine vasopressin as a contributor to a diuretic state.

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.

Effects of lung inflation on alveolar epithelial solute and water transport properties.

Paired hollow bullfrog lungs (Rana catesbeiana) were used to study the effects of lung inflation on alveolar epithelial transport of water and hydrophilic solutes. Frogs were double pithed and the lungs were removed after bronchial placement of a Lucite plug. Three openings in the plug accommodated the insertion of two agar-Ringer bridges (for electrical potential measurement and passage of direct current) and the injection and removal of alveolar bathing fluid. Ringer solution containing a tracer quantity of radioactive solute was instilled into the lung sacs (5 ml or 50 ml) and the lungs were suspended in baths of Ringer solution containng appropriate cold solutes (5 mM). Permeability properties of each solute (and water) were determined from the rate of radiotracer concentration change in the bath. The spontaneous potential difference, tissue resistance, and solute permeability properties determined in these experiments showed no significant differences between the 5- and 50-ml lungs. Assuming homogeneous, cylindrical water-filled pores to be present in the tissue, the equivalent pore radii estimated from the rates of solute and water fluxes were 1.1 (for 5-ml lungs) and 0.9 nm (for 50-ml lungs). After overinflation of the lung (to greater than 80 ml), experiments at 50 ml yielded a pore radius of 3.4 nm. These data suggest that passive alveolar epithelial transport properties do not change with degrees of lung inflation normally encountered in vivo but that overinflation can lead to increased leakiness of the barrier.

A model of epithelial water transport. The corneal endothelium

To try to understand how an epithelial tissue can transport water between bathing solutions of equal tonicity and how intracellular solute and protein concentration are related to the structural specialization of the cell membrane at its apical, basal, and lateral margins, we have formulated and solved, using approximate analytical techniques, a new model which combines the detailed transport of local osmotic flow in extracellular channel with the multicompartment approach of thermodynamic models requiring the overall conservation of water and solute for the entire cell layer. Thus, unlike most previous models, which dealt exclusively with either the average properties of the cell layer or the local transport in the extracellular channel, we are able to solve simultaneously for the interaction of the cell with its environments across its apical, basal, and lateral cell membranes as well as the detailed transport in the extracellular channel. The model is then applied to corneal endothelium to obtain new insight into the water flow movement in this tissue under in vitro and in vivo conditions. Then in vitro solution shows that the cell at 297 mosmol/liter is slightly hypotonic to the 300-mosmol/liter external bathing solutions which drive water equally out both the aqueous (apical) and stromal (basal) cell faces. This water is replaced from the extracellular channel. There is a net flow of water because more water enters the channel through its open stromal end than through the higher resistance tight junction. In vivo, the solution predicts that the stromal swelling pressure forces water through the tight junctions towards the stroma so that there is no net flow. The interesting new features of our solution are the water recirculation pattern and the role of the osmotically active proteins in making the cell hypertonic relative to the channel.

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.