The discovery of aquaporin-1 (AQP1) answered the long-standing biophysical question of how water specifically crosses biological membranes. In the kidney, at least seven aquaporins are expressed at distinct sites. AQP1 is extremely abundant in the proximal tubule and descending thin limb and is essential for urinary concentration. AQP2 is exclusively expressed in the principal cells of the connecting tubule and collecting duct and is the predominant vasopressin-regulated water channel. AQP3 and AQP4 are both present in the basolateral plasma membrane of collecting duct principal cells and represent exit pathways for water reabsorbed apically via AQP2. Studies in patients and transgenic mice have demonstrated that both AQP2 and AQP3 are essential for urinary concentration. Three additional aquaporins are present in the kidney. AQP6 is present in intracellular vesicles in collecting duct intercalated cells, and AQP8 is present intracellularly at low abundance in proximal tubules and collecting duct principal cells, but the physiological function of these two channels remains undefined. AQP7 is abundant in the brush border of proximal tubule cells and is likely to be involved in proximal tubule water reabsorption. Body water balance is tightly regulated by vasopressin, and multiple studies now have underscored the essential roles of AQP2 in this. Vasopressin regulates acutely the water permeability of the kidney collecting duct by trafficking of AQP2 from intracellular vesicles to the apical plasma membrane. The long-term adaptational changes in body water balance are controlled in part by regulated changes in AQP2 and AQP3 expression levels. Lack of functional AQP2 is seen in primary forms of diabetes insipidus, and reduced expression and targeting are seen in several diseases associated with urinary concentrating defects such as acquired nephrogenic diabetes insipidus, postobstructive polyuria, as well as acute and chronic renal failure. In contrast, in conditions with water retention such as severe congestive heart failure, pregnancy, and syndrome of inappropriate antidiuretic hormone secretion, both AQP2 expression levels and apical plasma membrane targetting are increased, suggesting a role for AQP2 in the development of water retention. Continued analysis of the aquaporins is providing detailed molecular insight into the fundamental physiology and pathophysiology of water balance and water balance disorders.

Aquaporins in the Kidney: From Molecules to Medicine

Water excretion by the kidney is regulated by the peptide hormone vasopressin. Vasopressin increases the water permeability of the renal collecting duct cells, allowing more water to be reabsorbed from collecting duct urine to blood. Despite long-standing interest in this process, the mechanism of the water permeability increase has remained undetermined. Recently, a molecular water channel (AQP-CD) has been cloned whose expression appears to be limited to the collecting duct. Previously, we immunolocalized this water channel to the apical plasma membrane (APM) and to intracellular vesicles (IVs) of collecting duct cells. Here, we test the hypothesis that vasopressin increases cellular water permeability by inducing exocytosis of AQP-CD-laden vesicles, transferring water channels from IVs to APM. Rat collecting ducts were perfused in vitro to determine water permeability and subcellular distribution of AQP-CD in the same tubules. The collecting ducts were fixed for immunoelectron microscopy before, during, and after exposure to vasopressin. Vasopressin exposure induced increases in water permeability and the absolute labeling density of AQP-CD in the APM. In parallel, the APM:IV labeling ratio increased. Furthermore, in response to vasopressin withdrawal, AQP-CD labeling density in the APM and the APM:IV labeling ratio decreased in parallel to a measured decrease in osmotic water permeability. We conclude that vasopressin increases the water permeability of collecting duct cells by inducing a reversible translocation of AQP-CD water channels from IVs to the APM.

https://www.pnas.org/content/pnas/92/4/1013.full.pdf

Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane

Two heme-peptides (HP) of about 20-A diameter (heme-undecapeptide [H11P], mol wt approximately 1900 and heme-octapeptide [H8P], mol wt approximately 1550), obtained by enzymic hydrolysis of cytochrome c, were sued as probe molecules in muscle capillaries (rat diaphragm). They were localized in situ by a perixidase reaction, enhanced by the addition of imidazole to the incubation medium. Chromatography of plasma samples showed that HPs circulate predominantly as monomers for the duration of the experiments and are bound by aldehyde fixatives to plasma proteins to the extent of approximately 50% (H8P) to approximately 95% (H11P). Both tracers cross the endothelium primarily via plasmalemmal vesicles which become progressively labeled (by reaction product) from the blood front to the tissue front of the endothelium, in three successive resolvable phases. By the end of each phase the extent of labeling reaches greater than 90% of the corresponding vesicle population. Labeled vesicles appear as either isolated units or chains which form patent channels across the endothelium. The patency of these channels was checked by specimen tilting and graphic analysis of their images. No evidence was found for early or preferential marking of the intercellular junctions and spaces by reaction product. It is concluded that the channels are the most likely candidate for structural equivalents of the small pores of the capillary wall since they are continuous, water-filled passages, and are provided with one or more strictures of less than 100 A. Their frequency remains to be established by future work.

/pdf/permeability-of-muscle-capillaries-to-small-heme-peptides-27eychoh59.pdf

Permeability of muscle capillaries to small heme-peptides. Evidence for the existence of patent transendothelial channels.

It is now well established that the action of antidiuretic hormone (ADH) is mediated by cAMP and that ADH induces physiological changes in the luminal cell Less certain at this stage are the events that link an elevation of intracellular cAMP to an increase in luminal membrane permeability to water and solutes. In recent years, three observations on the toad bladder have provided important insights into these post-CAMP events: (1) The demonstration that by a variety of maneuvers the changes in water permeability can be dissociated from changes in solute Permeability and therefore that separate pathways are involved.5-7 (2) The observations demonstrating that microtubules and microfilaments are involved in the water permeability response.8-10 (3 ) The finding that intramembrane particle aggregates visualized by freeze-fracture electron microscopy are associated with the modulation of luminal-membrane water pem1eabi1ity.ll-l~ Observations reported by many laboratories clearly demonstrate that these organized membrane structures perform an important role in the action of ADH.ll-la Most workers in the field now strongly suspect that these structures are the site of transmembrane water channels. The evidence that aggregates play a role in the ADH response has been discussed in several review papers.l73 Here we will describe how freeze-fracture observations have led to a new proposal for the mechanism of action of ADH and discuss to what extent this proposal is supported by the available evidence.

ADH Action: Evidence for a Membrane Shuttle Mechanism

The Mercurial Insensitive Water Channel (AQP-4) Forms Orthogonal Arrays in Stably Transfected Chinese Hamster Ovary Cells

Freeze-fracture electron microscopy demonstrates that vasopressin stimulation of isolated toad bladder alters the structure of the luminal membrane of granular cells. This alteration consists of an ordered aggression of intramembranous particles, and appears to be of functional significance, since the frequency of aggregation sites per area of membrane is closely correlated with vasopressin-induced osmotic water flow.

Vasopressin: induced structural change in toad bladder luminal membrane

Freeze-fracture electronmicroscopy demonstrates that vasopressin stimulation of isolated toad bladder results in a striking morphologic alteration of epithelial membrane structure. This alteration is characterized by the aggregation of intramembranous particles in orderly linear arrays at multiple sites in the luminal membranes of granular cells specifically. The size of these aggregates varies considerably, in terms of area, over a range from 0.5 to 70×10−3 μm2. The median aggregate size is about 10.5×10−3 μm2. Since the extent of vasopressin-associated particle aggregation, in terms of frequency of sites per area of membrane or cumulative area of membrane occupied by them, closely correlates with induced changes in transport function, as measured by osmotic water flow, the aggregates themselves appear to be of physiologic significance in the mechanism of action of vasopressin. This hypothesis is supported by the observations that sites of aggregation occur (a) in response to serosal exposure to hormone specifically, (b) independently of an osmotic gradient, and (c) following stimulation with cyclic adenosine monophosphate.

Membrane structural and functional responses to vasopressin in toad bladder

Numerous studies have suggested that the lateral intercellular spaces can serve as part of the pathway for water movement across epithelia (2, 3, 5, 6, 11, 14, 15, 20, 22, 23, 24) . The primary evidence for this view has been the frequent observation in several tissues that increases in net fluid transport is accompanied by marked widening of intercellular spaces. However, recent work has demonstrated that widened intercellular spaces can occur without net water transfer, possibly due to contractile activity of smooth muscle (8, 9) . It has also been observed that the degree of stretch imposed on tissue can markedly affect the width of intercellular spaces (9) . Thus, it would be useful to follow water movement through intercellular spaces by a technique other than size changes .

/pdf/the-effect-of-osmotic-flow-on-the-distribution-of-1vuca5pv4o.pdf

James B. Wade

Papers

Vasopressin: induced structural change in toad bladder luminal membrane

Membrane structural and functional responses to vasopressin in toad bladder

The effect of osmotic flow on the distribution of horseradish peroxidase within the intercellular spaces of toad bladder epithelium.