Permeability For Small Solutes Research Articles

Tail-Oxidized Cholesterol Enhances Membrane Permeability for Small Solutes.

Cholesterol renders mammalian cell membranes more compact by reducing the amount of voids in the membrane structure. Because of this, cholesterol is known to regulate the ability of cell membranes to prevent the permeation of water and water-soluble molecules through the membranes. Meanwhile, it is also known that even seemingly tiny modifications in the chemical structure of cholesterol can lead to notable changes in membrane properties. The question is, how significantly do these small changes in cholesterol structure affect the permeability barrier function of cell membranes? In this work, we applied fluorescence methods as well as atomistic molecular dynamics simulations to characterize changes in lipid membrane permeability induced by cholesterol oxidation. The studied 7β-hydroxycholesterol (7β-OH-chol) and 27-hydroxycholesterol (27-OH-chol) represent two distinct groups of oxysterols, namely, ring- and tail-oxidized cholesterols, respectively. Our previous research showed that the oxidation of the cholesterol tail has only a marginal effect on the structure of a lipid bilayer; however, oxidation was found to disturb membrane dynamics by introducing a mechanism that allows sterol molecules to move rapidly back and forth across the membrane—bobbing. Herein, we show that bobbing of 27-OH-chol accelerates fluorescence quenching of NBD-lipid probes in the inner leaflet of liposomes by dithionite added to the liposomal suspension. Systematic experiments using fluorescence quenching spectroscopy and microscopy led to the conclusion that the presence of 27-OH-chol increases membrane permeability to the dithionite anion. Atomistic molecular dynamics simulations demonstrated that 27-OH-chol also facilitates water transport across the membrane. The results support the view that oxysterol bobbing gives rise to successive perturbations to the hydrophobic core of the membrane, and these perturbations promote the permeation of water and small water-soluble molecules through a lipid bilayer. The observed impairment of permeability can have important consequences for eukaryotic organisms. The effects described for 27-OH-chol were not observed for 7β-OH-chol which represents ring-oxidized sterols.

Autophagy and tight junction proteins in the intestine and intestinal diseases.

The intestinal epithelium (IE) forms an indispensible barrier and interface between the intestinal interstitium and the luminal environment. The IE regulates water, ion and nutrient transport while providing a barrier against toxins, pathogens (bacteria, fungi and virus) and antigens. The apical intercellular tight junctions (TJ) are responsible for the paracellular barrier function and regulate trans-epithelial flux of ions and solutes between adjacent cells. Increased intestinal permeability caused by defects in the IE TJ barrier is considered an important pathogenic factor for the development of intestinal inflammation, diarrhea and malnutrition in humans and animals. In fact, defects in the IE TJ barrier allow increased antigenic penetration, resulting in an amplified inflammatory response in inflammatory bowel disease (IBD), necrotizing enterocolitis and ischemia-reperfusion injury. Conversely, the beneficial enhancement of the intestinal TJ barrier has been shown to resolve intestinal inflammation and apoptosis in both animal models of IBD and human IBD. Autophagy (self-eating mechanism) is an intracellular lysosome-dependent degradation and recycling pathway essential for cell survival and homeostasis. Dysregulated autophagy has been shown to be directly associated with many pathological processes, including IBD. Importantly, the crosstalk between IE TJ and autophagy has been revealed recently. We showed that autophagy enhanced IE TJ barrier function by increasing transepithelial resistance and reducing the paracellular permeability of small solutes and ions, which is, in part, by targeting claudin-2, a cation-selective, pore-forming, transmembrane TJ protein, for lysosome (autophagy)-mediated degradation. Interestingly, previous studies have shown that the inflamed intestinal mucosa in patients with active IBD has increased claudin-2 expression. In addition, inflammatory cytokines (for example, tumor necrosis factor-α, interleukin-6, interleukin-13, and interleukin-17) whose levels are increased in IBD patients cause an increase in claudin-2 expression and a claudin-2-dependent increase in TJ permeability. Thus, the role of claudin-2 in intestinal pathological processes has been attributed, in part, to the increase of intestinal TJ permeability. Claudin-2 represents a new therapeutic target in treating IBD, diarrhea and malnutrition in animals and humans.

Molecular Dynamics Simulation of Inverse-Phosphocholine Lipids

We have performed molecular dynamics simulations of lipid bilayers composed of inverse-phosphatidylcholines (CPe), an analog of phosphatidylcholine (PC) with the choline group directly bound to the glycerol backbone and the phosphate group freely protruding into the water phase. Synthetic phospholipids with the CPe headgroup have been proposed for use as drug delivery liposomes. Our simulation results show that the CPe lipids were characterized by a larger area per lipid molecule than the PC lipids. This can partly explain experimental results that show a higher permeability of small solutes through the membranes of liposomes composed of them. Unlike the PC headgroup, the CPe headgroup was found not to bind sodium ions at the water membrane interface. Both lipid types were found to bind calcium ions but do not bind potassium ions. Inversion of the choline group was found to decrease hydration of the membrane in the carbonyl region of the bilayer as well as hydration of the choline group. From analyzing the water ordering in our simulation, we determined that the orientation of the water layer next to the CPe membrane is effectively inverted with respect to the water ordering of the PC membrane, possibly affecting interaction with biomembranes encountered in drug delivery. Due to changes in ion binding, charge group distribution, and water orientation, the electrostatic potential profiles across the lipid bilayer of CPe membranes were found to differ considerably from those of PC membranes. This is a possible explanation of the experimentally observed changes in the charged solute permeability.

Aquaporin-1: New Developments and Perspectives for Peritoneal Dialysis

Peritoneal dialysis involves diffusive and convective transport and osmosis through the highly vascularized peritoneal membrane. Several lines of evidence have demonstrated that the water channel aquaporin-1 (AQP1) corresponds to the ultrasmall pore predicted by the model of peritoneal transport. Proof-of-principle studies have shown that upregulation of the expression of AQP1 in peritoneal capillaries results in increased water permeability and ultrafiltration, without affecting the osmotic gradient or small solute permeability. Conversely, studies in Aqp1 mice have shown that haplo-insufficiency for AQP1 results in significant attenuation of water transport. Recent studies have demonstrated that AQP1 is involved in the migration of different cell types, including endothelial cells. In parallel, chemical screening has identified lead compounds that could act as antagonists or agonists of AQPs, with description of putative binding sites and potential mechanisms of gating the water channel. By modulating water transport, these pharmacological agents could have clinically relevant effects in targeting specific tissues or disease states.

A Pathway Separate from the Central Channel through the Nuclear Pore Complex for Inorganic Ions and Small Macromolecules

Nuclear pore complexes (NPCs) are supramolecular nanomachines that mediate the exchange of macromolecules and inorganic ions between the nucleus and the cytosol. Although there is no doubt that large cargo is transported through the centrally located channel, the route of ions and small molecules remains debatable. We thus tested the hypothesis that there are two separate pathways by imaging NPCs using atomic force microscopy, NPC electrical conductivity measurements, and macromolecule permeability assays. Our data indicate a spatial separation between the active transport of macromolecules through the central channel and the passive transport of ions and small macromolecules through the pore periphery.

TRPV4‐mediated regulation of epithelial permeability

TRPV4 is a calcium-permeable channel activated by extracellular hypotonicity, polyunsaturated fatty acids, phorbol esters, and heat. We show that TRPV4 is localized in the basolateral membrane of the mouse mammary cell line HC11. Activation of TRPV4 caused an increase in the intracellular Ca(2+) concentration through influx of extracellular Ca(2+), triggering two independent chains of events: 1) a rapid increase in transcellular conductance through the activation of apical large conductance calcium-activated (BK) potassium channels that were blockable by paxilline; 2) a slow increase in paracellular permeability for small solutes. The latter effect was accompanied by a down-regulation of the tight junctional proteins claudin -1, -3, -4, -5, -7, and -8 and by dramatic changes in tight junction morphology, including frequent large breaks in the tight junction strands. This dual modulation of epithelial permeability after TRPV4 activation may be involved in regulating the tonicity across mammary gland epithelia. TRPV4 activation may also be responsible for exudation and edema formation during inflammation processes.-Reiter, B., Kraft, R., Günzel, D., Zeissig, S., Schulzke, J-D., Fromm, M., Harteneck, C. TRPV4-mediated regulation of epithelial permeability.

Aquaporin‐1 in the peritoneal membrane: implications for peritoneal dialysis and endothelial cell function

PD (peritoneal dialysis) is an established mode of renal replacement therapy, based on the exchange of fluid and solutes between blood in peritoneal capillaries and a dialysate that has been introduced into the peritoneal cavity. The dialysis process involves diffusive and convective transports and osmosis through the PM (peritoneal membrane). Computer simulations predicted that the PM contains ultrasmall pores (radius <3 A, 1 A=10(-10) m), responsible for up to 50% of UF (ultrafiltration), i.e. the osmotically driven water movement during PD. Several lines of evidence suggest that AQP1 (aquaporin-1) is the ultrasmall pore responsible for transcellular water permeability during PD. Treatment with corticosteroids induces the expression of AQP1 in the PM and improves water permeability and UF in rats without affecting the osmotic gradient and permeability for small solutes. Studies in knockout mice provided further evidence that osmotically driven water transport across the PM is mediated by AQP1. AQP1 and eNOS (endothelial nitric oxide synthase) show a distinct regulation within the endothelium lining the peritoneal capillaries. In acute peritonitis, the up-regulation of eNOS and increased release of nitric oxide dissipate the osmotic gradient and prevent UF, whereas AQP1 expression is unchanged. These results illustrate the usefulness of the PM to investigate the role and regulation of AQP1 in the endothelium. The results also emphasize the critical role of AQP1 during PD and suggest that manipulation of AQP1 expression may be used to increase water permeability across the PM.

Lung Fluid Balance During Development

After completing this article, readers should be able to: 1. Describe the major driving forces for liquid formation in and liquid removal from the lumen of the fetal lung. 2. Delineate the role of a normal volume of liquid within the lumen of the fetal lung for normal lung growth during development. 3. Describe the effects of labor on Na,K-ATPase activity in lung epithelial cells and clearance of lung liquid after birth. 4. Explain the route by which most residual liquid drains from the lung. 5. List the conditions associated with preterm birth that may contribute to delayed removal of fetal lung liquid. Curious students of mammalian development recognized more than a century ago that the lungs are filled with liquid during fetal life, (1) but the origin of that liquid remained obscure until 1948, when an unexpected discovery by two French scientists dispelled the prevailing view that fetal lung liquid derived from intrauterine inhalation of amniotic fluid. In studies designed to explore the ontogeny of the pituitary-adrenal axis, tracheal ligation of fetal rabbits for 9 days resulted in fluid distention of the lungs. (2) This serendipitous finding, subsequently confirmed and embellished by others, (3)(4)(5) established that the fetal lung itself, rather than the amniotic sac, is the source of the liquid that fills the lung during development. This liquid forms a slowly expanding structural template that prevents collapse and promotes growth of the fetal lung. Subsequent studies, conducted mostly with sheep, showed that liquid in the lumen of the fetal lung forms as a result of chloride secretion in the respiratory epithelium, (6)(7) a process that can be inhibited by diuretics that block Na,K-2Cl cotransport. (8)(9) In vitro experiments using cultured explants of lung tissue and monolayers of epithelial cells harvested from human fetal lung have …

Inhibition of Nitric Oxide Synthase Reverses Permeability Changes in a Mouse Model of Acute Peritonitis

Acute peritonitis is the most frequent complication of peritoneal dialysis. Previous studies have suggested a major role for nitric oxide (NO) in the permeability changes and loss of ultrafiltration induced by acute peritonitis. In this study, we further investigated the potential role of NO in a mouse model of peritonitis induced by Escherichia coli Lipopolysaccharide (LPS). A 2-hour peritoneal equilibration test was performed in control and LPS-treated mice using 7% glucose dialysate supplemented or not with the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). The levels of NO metabolites in the dialysate were maximal 18 hours after LPS injection. At that time, acute peritonitis induced by LPS was reflected by an increased recruitment of leukocytes, an increased intraperitoneal release of interleukin-6, a significant increase in the peritoneal permeability for small solutes, a loss of sodium sieving, and a loss of ultrafiltration in comparison with controls. Addition of L-NAME in LPS-treated mice significantly reversed permeability modifications and prevented the release of NO metabolites into the dialysate. These results confirm that increased NO mediates permeability modifications during acute peritonitis, and illustrate the potential of mouse models to investigate the molecular mechanisms regulating peritoneal permeability.

Molecular mechanisms modifying the peritoneal membrane exposed to peritoneal dialysis.

Over time, a significant proportion of patients an peritoneal dialysis (PD) develop an increased permeability for small solutes, which induces a faster absorption of glucose, and ultrafiltration failure by early dissipation of the osmotic gradient. Vascular proliferation and vasodilatation of preexisting vessels might represent the structural basis for increased effective peritoneal surface area encountered in these PD patients. Animal models have shown that local release of growth factors and nitric oxide in the peritoneal membrane (PM) may lead to the development of areas of neovascularization and/or submesothelial fibrosis. Long-term exposure to conventional, glucose-based dialysis fluids plays a central role in the pathogenesis of these structural modifications. Glucose degradation products and reactive carbonyl species, which are present both in glucose-based dialysates and uremic plasma, accelerate the formation of the advanced glycation end products in the PM, which may in turn initiate a range of cellular responses including stimulation of monocytes, secretion of inflammatory cytokines, proliferation of vascular smooth muscle cells, stimulation of growth factors, and secretion of matrix proteins. The changes in the PM may also be potentiated by uremia and hyperglycemia per se. These new insights into the molecular mechanisms operating in the PM have provided rationale for novel therapeutic strategies including the development of glucose-free PD solutions and two-chamber bags.

Corticosteroids induce expression of aquaporin-1 and increase transcellular water transport in rat peritoneum.

The water channel aquaporin-1 (AQP1) is the molecular counterpart of the ultrasmall pore responsible for transcellular water permeability during peritoneal dialysis (PD). This water permeability accounts for up to 50% of ultrafiltration (UF) during a hypertonic dwell, and its loss can be a major clinical problem for PD patients. By analogy with the lung, the hypothesis was tested that corticosteroids may increase AQP1 expression in the peritoneal membrane (PM) and improve water permeability and UF in rats. First, the expression and distribution of the glucocorticoid receptor (GR) in the PM and capillary endothelium was documented. Time-course and dose-response analyses showed that a daily IM injection of dexamethasone (1 or 4 mg/kg) for 5 d induced an approximately twofold increase in the expression of AQP1 at the mRNA and protein levels. The GR antagonist RU-486 completely inhibited the dexamethasone effect. The functional counterpart of the increased AQP1 expression was a significant increase in sodium sieving and net UF across the PM, contrasting with a lack of effect on the osmotic gradient and permeability for small solutes. The latter observation reflected the lack of effect of corticosteroids on nitric oxide synthase (NOS) activity and endothelial NOS isoform expression in the PM. In conclusion, corticosteroids induce AQP1 expression in the capillary endothelium of the PM, which is reflected by increased transcellular water permeability and UF. These data emphasize the critical role of AQP1 during PD and suggest that pharmacologic regulation of AQP1 may provide a target for manipulating water permeability across the PM.

Experimental diabetes induces functional and structural changes in the peritoneum

Peritoneal dialysis (PD) is an established renal replacement therapy in diabetic patients, but the influence of diabetes on the peritoneal membrane (PM) remains debated. We have used functional, biochemical and molecular studies in vivo and in vitro to substantiate the changes induced by diabetes and hyperglycemia in the PM. Peritoneal equilibration tests were performed 2, 4, and 6 weeks after induction of diabetes with streptozotocin (STZ) in rats. Morphological analyses, determination of nitric oxide synthase (NOS) activities, and expression studies for NOS isoforms and advanced glycation end products (AGE) were performed in parallel. Additional studies were conducted in diabetic rats treated with insulin, non-diabetic rats fed with urea, and cultured bovine aortic endothelial cells (BAEC). In comparison with controls, diabetic rats were characterized by: increased permeability for small solutes and decreased sodium sieving; capillary proliferation; increased endothelial NOS (eNOS) and AGE immunoreactivity; up-regulation of eNOS and down-regulation of neuronal NOS; and increased NOS activity in the PM. The changes, which culminated at week 6, were prevented by chronic insulin treatment in diabetic rats. In contrast to hyperglycemia, hyperosmolality alone did not induce functional or structural changes in the PM. Studies in BAEC showed that high glucose incubation led to increased activity and expression of eNOS, a prerequisite for vascular proliferation. These data demonstrate that chronic hyperglycemia is associated with functional and structural changes in the peritoneum that parallel with selective regulation of NOS isoforms and AGE deposits. The alterations are prevented by insulin treatment, which suggests that adequate control of diabetes can preserve PM integrity in diabetic patients prior to PD.

Molecular Mechanisms of Peritoneal Permeability— Research in Growth Factors

eritoneal dialysis (PD) is now an established treatment of end-stage renal failure, used in approximately 15% of dialysis patients worldwide. Over the past few years, the morbidity associated with acute peritonitis has been considerably reduced (1). The major problem remaining with long-term PD is the high incidence of ultrafiltration (UF) failure, which can affect up to 50% of PD patients treated for more than 6 years (2,3). Cross-sectional and longitudinal studies have shown that peritoneal solute transport increases with time on PD (4). This increased permeability for small solutes means faster absorption of glucose, early dissipation of the osmotic gradient, and, eventually, UF failure (5). The relevance of the modifications in the peritoneal membrane is illustrated by the fact that high peritoneal membrane permeability is a significant risk factor for PD patients, predicting both technical failure and patient death (6). This brief review discusses how structural, functional, and molecular data have improved our understanding of the modifications to the peritoneal membrane in long-term PD. STRUCTURAL MODIFICATIONS TO THE PERITONEUM

Inhibition of nitric oxide synthase reverses changes in peritoneal permeability in a rat model of acute peritonitis

Acute peritonitis is the most frequent complication of peritoneal dialysis (PD), and nitric oxide (NO) is thought to play a role in the structural and permeability changes observed in this condition. We have used a combination of expression, enzymatic and pharmacological studies to substantiate the potential role(s) played by NO during peritonitis. The peritoneal equilibration test was performed in control rats and rats with acute peritonitis (originating from skin flora), using standard dialysate supplemented or not with the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). In parallel, peritoneal NOS enzymatic activities were measured and expression studies for NOS isoforms and S-nitrosocysteine reactivity performed in the peritoneum. In comparison with controls, rats with acute peritonitis were characterized by inflammatory changes, increased S-nitrosocysteine immunoreactivity, and increased NOS activities in the peritoneum, due to the up-regulation of endothelial and inducible NOS. In parallel, rats with acute peritonitis showed increased permeability for small solutes; decreased sodium sieving; loss of ultrafiltration (UF); and increased protein loss in the dialysate. Addition of L-NAME to the dialysate did not induce permeability changes in control rats, but significantly improved UF and reversed permeability modifications in rats with peritonitis. The effect of L-NAME was reflected by a mild but consistent increase in blood pressure during PD exchange. Our results demonstrate that local generation of NO, secondary to up-regulation of NOS isoforms, plays an important role in the regulation of peritoneal permeability during acute peritonitis in rats. By itself, NOS inhibition improves UF and reverses permeability changes, which might offer new therapeutic perspectives in acute peritonitis.

Chronic Uremia Induces Permeability Changes, Increased Nitric Oxide Synthase Expression, and Structural Modifications in the Peritoneum

Advanced glycation end products (AGE), growth factors, and nitric oxide contribute to alterations of the peritoneum during peritoneal dialysis (PD). These mediators are also involved in chronic uremia, a condition associated with increased permeability of serosal membranes. It is unknown whether chronic uremia per se modifies the peritoneum before PD initiation. A rat model of subtotal nephrectomy was used to measure peritoneal permeability after 3, 6, and 9 wk, in parallel with peritoneal nitric oxide synthase (NOS) isoform expression and activity and structural changes. Uremic rats were characterized by a higher peritoneal permeability for small solutes and an increased NOS activity due to the up-regulation of endothelial and neuronal NOS. The permeability changes and increased NOS activities correlated with the degree of renal failure. Focal areas of vascular proliferation and fibrosis were detected in uremic rats, in relation with a transient up-regulation of vascular endothelial growth factor and basic fibroblast growth factor, as well as vascular deposits of the AGE carboxymethyllysine and pentosidine. Correction of anemia with erythropoietin did not prevent the permeability or structural changes in uremic rats. Thus, in this rat model, uremia induces permeability and structural changes in the peritoneum, in parallel with AGE deposits and up-regulation of specific NOS isoforms and growth factors. These data suggest an independent contribution of uremia in the peritoneal changes during PD and offer a paradigm to better understand the modifications of serosal membranes in uremia.

Arterial hypertension increases intracranial pressure in cat after opening of the blood-brain barrier.

Increased permeability for small solutes in brain capillaries means that a change in hydrostatic capillary pressure may influence transcapillary fluid exchange according to the Starling fluid equilibrium, and a high arterial pressure may cause transcapillary fluid filtration and raised intracranial pressure. This could be of clinical relevance in states of disrupted blood-brain barrier such as meningitis and after a severe head injury, especially since these patients quite often are spontaneously hypertensive, and hypertensive therapy is sometimes used to increase cerebral perfusion pressure. This study on cat investigated the long-term relation between arterial pressure and intracranial pressure in a state of disrupted blood-brain barrier. Endotoxin was given intrathecally to open the blood-brain barrier and depress cerebral autoregulation. Arterial pressure was increased by about 30 mm Hg during 5 hours by dopamine and angiotensin II infusion. The immediate fall in intracranial pressure after normalization of blood pressure reflects the blood volume component of an intracranial pressure increase. Increased arterial pressure had no effect on intracranial pressure before endotoxin. Endotoxin infusion increased intracranial pressure from the normal value of 10 to 12 mm Hg. and at steady state by almost 10 mm Hg. Intracranial pressure increased further after the arterial pressure increase. At steady state (achieved within 5 hours), this increase was almost as great as the arterial pressure increase, and about 80% persisted when measured directly after normalization of the arterial pressure. Increased arterial pressure in a state of disrupted blood-brain barrier increases intracranial pressure, mainly because of brain edema. This stresses that arterial hypertension may be deleterious in conditions such as meningitis or after a brain trauma.

Role of cadherins 5 and 13 in the aortic endothelial barrier.

We investigated the role of the cadherins 5 and 13 in the solute barrier formed by aortic endothelial cells in vitro. In confluent monolayers of bovine aortic endothelial cells, immunofluorescence with antibodies to the external domain of cadherin 5 (Mab 9H7) or to cadherin 13 (Mab Ec6C10) found staining for both cadherins at endothelial cell borders. Western blotting with an antibody to the characteristic cadherin cytoplasmic tail or with an antibody to the extracellular domain of cadherin 5 revealed a single 125 kD protein band. A second larger band was found at 130 kD with the anti-cadherin 13 Mab which was not recognized by an antibody to the cadherin cytoplasmic tail. A calcium switch strategy was used to investigate the involvement of these cadherins in the endothelial barrier. Changes in the permeability of small solutes in an endothelial cell column produced by a decrease in calcium concentration followed by a return to normal calcium, with or without antibody, were recorded. We found that anti-cadherin 5 IgG (10 micrograms/ml) interfered with the reforming of interendothelial junctions after restoration of calcium at every time point tested for a total of 45 min after restoration of calcium. The anti-cadherin 13 IgG (10 micrograms/ml) did not block reforming of the endothelial barrier in a similar manner. The presence of this antibody delayed only by 15 min the restoration of the normal barrier. Without calcium switch, addition of either monoclonal antibody (10 micrograms/ml) to the endothelial cell column had no effect on solute permeability. These results suggest that cadherin 5 in bovine aortic endothelial cells has a major functional role in forming the calcium-sensitive endothelial junction in vitro and may play an important role in the normal structure and function of the in vivo barrier.

In vivo inhibition of transcellular water channels (aquaporin-1) during acute peritoneal dialysis in rats.

During peritoneal dialysis (PD), a major portion of the osmotically induced water transport to the peritoneum can be predicted to occur through endothelial water-selective channels. Aquaporin-1 (AQP-1) has recently been recognized as the molecular correlate to such channels. Aquaporins can be inhibited by mercurials. In the present study, HgCl2 was applied locally to the peritoneal cavity in rats after short-term tissue fixation, used to protect the tissues from HgCl2 damage. Dianeal (3.86%) was employed as dialysis fluid, 125I-albumin as an intraperitoneal volume marker, and 51Cr-EDTA (constantly infused intravenously) to assess peritoneal small-solute permeability characteristics. Immunocytochemistry and immunoelectron microscopy revealed abundant AQP-1 labeling in capillary endothelium in peritoneal tissues, representing sites for HgCl2 inhibition of water transport. HgCl2 treatment reduced water flow and inhibited the sieving of Na+ without causing any untoward changes in microvascular permeability, compared with that of fixed control rats, in which the peritoneal cavity was exposed to tissue fixation alone. In fixed control rats, the mean intraperitoneal volume (IPV) increased from 20.5 +/- 0.15 to 25.0 +/- 0.52 ml in 60 min, whereas in the HgCl2-treated rats, the increment was only from 20.7 +/- 0.23 to 23.5 +/- 0.4 ml. In fixed control rats, the dialysate Na+ fell from 135.3 +/- 0.97 to 131.3 +/- 1.72 mM, whereas in the HgCl2-treated rats the dialysate Na+ concentration remained unchanged between 0 and 40 min, further supporting that water channels had been blocked. Computer simulations of peritoneal transport were compatible with a 66% inhibition of water flow through aquaporins. The observed HgCl2 inhibition of transcellular water channels strongly indicates a critical role of aquaporins in PD and provides evidence that water channels are crucial in transendothelial water transport when driven by crystalloid osmosis.

Permeability of cat skeletal muscle capillaries to small solutes.

Permeability-surface area products (PS) for 51Cr-EDTA, [3H]mannitol, [14C]urea, and 22Na were measured in isolated, perfused, lower hindlimb muscles of anesthetized cats. The tracers were added stepwise to the arterial inflow, and Evans blue-labeled albumin was the reference indicator. At flow rates > 70 ml.min-1.100 g-1, the PS values (+/- SE) were 5.0 +/- 0.5, 7.6 +/- 1.2, 17.8 +/- 1.4, and 21.0 +/- 1.4 (n = 7, 4, 5, and 3 animals, respectively). The ratio of simultaneous PS measurements of mannitol and urea was 0.42 +/- 0.02 (n = 9), significantly less than the free diffusion coefficient ratio (0.49), indicating the presence of restricted diffusion. PS measurements were also made during osmotic flow (4.2 +/- 0.6 ml.min-1.100 g-1) induced by 20% NaCl. The data clearly showed that osmotic transients did not alter small solute permeability. Pore models were used to show that the PS data and previously reported reflection coefficient data were consistent with a single description of the capillary wall. This model contained a water-only pathway containing 60% of the hydraulic capacity and an extracellular route modeled by pores of 4 nm radius having 21,000 cm of area per unit membrane thickness (A/delta x).

Microcirculatory structure-function relationships in skeletal muscle of diabetic rats.

The effects of streptozotocin-induced diabetes on microcirculatory structure-function relationships in skeletal muscle were studied in control (C) and diabetic (D; 65 mg/kg streptozotocin ip) rats 6-8 wk after injection. Capillary exchange capacity was determined from measurements of capillary filtration coefficient (CFC) and permeability-surface area product (PS) for 51Cr-labeled EDTA in maximally vasodilated (papaverine), isolated hindquarters of C (n = 9) and D (n = 12) rats. Capillary numerical density, length, surface area, capillary geometry, and muscle fiber cross-sectional area were determined using morphometric methods in perfusion-fixed plantaris muscles from a second series of C (n = 5) and D (n = 6) rats. Hindquarters of D rats (61 +/- 3 g) weighed less than C rats (90 +/- 3 g) because of marked muscle atrophy. Minimal total vascular resistance was lower in D rats (P < or = 0.05), indicating an increased flow capacity. CFC was not different in C and D rats (0.0282 +/- 0.0020 vs. 0.0330 +/- 0.0025 ml.min-1.mmHg-1 x 100 g-1, respectively). The relationship between PS and flow was depressed in D rats (P < or = 0.05) compared with C rats, which indicated a reduced capillary diffusing capacity. Plantaris muscle weight was 41% less in D rats (174 +/- 9 vs. 293 +/- 11 mg; P < or = 0.001). Morphometric analysis revealed that muscle fiber cross-sectional area was reduced 39% in D rats, which, despite a lower capillary-to-fiber ratio (1.59 +/- 0.04 vs. 2.12 +/- 0.13; P < or = 0.001), resulted in a 27% increase in capillary density in D rats. Capillary diameter was less in D rats (3.58 +/- 0.12 vs. 4.51 +/- 0.23 microns; P < or = 0.005). Total capillary surface area was reduced 42% in D rats; however, capillary surface area per muscle fiber volume was unchanged in D rats (231 +/- 34 vs. 237 +/- 16 cm-1). These data indicate that there is remodeling of the capillary bed in skeletal muscle of D rats, resulting in a reduction in total microvascular surface area. The reduction in capillary surface area is proportional to the degree of muscle atrophy in D rats such that functional microvascular surface area per tissue mass (e.g., CFC) is unchanged. The lower diffusing capacity (PS) in D rats suggests that either small solute permeability is reduced and/or there is greater perfusion heterogeneity in D rat skeletal muscle.