Glomerular endothelium: a porous sieve and formidable barrier

The glomerular capillary endothelium is highly specialized to support the selective filtration of massive volumes of plasma. Filtration is driven by Starling forces acting across the glomerular capillary wall, and depends on its large surface area and extremely high water permeability. Glomerular endothelial cells are extremely flat and perforated by dense arrays of trans-cellular pores, the fenestrae. This phenotype is critical for the high glomerular water permeability and depends on podocyte-derived VEGF, as well as TGF-beta. Endothelial cell-derived PDGFB, in turn, is necessary for the establishment of mesangial cells, which sculpt the glomerular loop structure that underlies the large filtration surface area. In pre-eclampsia, inhibition of the VEGF- and TGF-beta signaling pathways leads to endothelial swelling and loss of fenestrae, reducing the glomerular filtration rate. Similarly, in the thrombotic microangiopathies, glomerular endothelial cell injury coupled with inappropriate VWF activation leads to intracapillary platelet aggregation and loss of the flat, fenestrated phenotype, thus reducing the glomerular filtration rate. Normally, a remarkably small fraction of albumin and other large plasma proteins passes across the glomerular capillary wall despite the massive filtration of water and small solutes. An elaborate glycocalyx, which covers glomerular endothelial cells and their fenestrae forms an impressive barrier that, together with other components of the glomerular capillary wall, prevents loss of plasma proteins into the urine. Indeed, microalbuminuria is a marker for endothelial glycocalyx disruption, and most forms of glomerular endothelial cell injury including pre-eclampsia and thrombotic microangiopaties can cause proteinuria.     

Fluorescence correlation spectroscopy can probe albumin dynamics inside lung endothelial glycocalyx

The endothelial glycocalyx is believed to play a major role in capillary permeability by functioning as a macromolecular barrier overlying the intercellular junction. Little is known about the functional attributes of the glycocalyx (i.e., porosity and permeability) or which constituents contribute to its overall structure-function relationship. In this report, we demonstrate the utility of fluorescence correlation spectroscopy (FCS) to measure albumin diffusion rates and concentration profiles above the cell surface and overlying the intercellular junctions of lung capillary endothelial cells. Albumin diffusion rates and concentration profiles were obtained before and after enzymatic digestion of the glycocalyx with pronase, heparanase, or hyaluronidase. The results suggest a structure interacting with albumin located from 1.0 to 2.0 microm above the cell membrane capable of reducing albumin diffusion by 30% while simultaneously increasing albumin concentration fivefold. Digestion of the glycocalyx with pronase or heparanase resulted in only modest changes in albumin diffusion and concentration profiles. Hyaluronidase digestion completely eliminated albumin-glycocalyx interactions. These data also suggest that hyaluronan is a major determinant for albumin interactions with the lung endothelial glycocalyx. Confocal images of heparan sulfate and hyaluronan confirm a cell-surface layer 2-3 mum in thickness, thus supporting FCS measurements. In summary, we report the first use of FCS to probe extracellular structures and further our understanding of the structure-function relationship of the lung microvascular endothelial glycocalyx.     

Determination of microvessel permeability and tissue diffusion coefficient of solutes by laser scanning confocal microscopy

Interstitium contains a matrix of fibrous molecules that creates considerable resistance to water and solutes in series with the microvessel wall. On the basis of our preliminary studies, by using laser-scanning confocal microscopy and a theoretical model for interstitial transport, we determined both microvessel solute permeability (P) and solute tissue diffusion coefficient (D) of alpha-lactalbumin (Stokes radius 2.01 nm) from the rate of tissue solute accumulation and the radial concentration gradient around individually perfused microvessel in frog mesentery. P(alpha-lactalbumin) is 1.7 +/- 0.7(SD) x 10(-6) cm/s (n = 6). D(t)/D(free) for alpha-lactalbumin is 27% +/- 5% (SD) (n = 6). This value of D(t)/D(free) is comparable to that for small solute sodium fluorescein (Stokes radius 0.45 nm), while p(alpha-lactalbumin) is only 3.4% of p(sodium fluorescein). Our results suggest that frog mesenteric tissue is much less selective to solutes than the microvessel wall.     

Starling forces that oppose filtration after tissue oncotic pressure is increased

We tested the hypothesis that the effective oncotic force that opposes fluid filtration across the microvessel wall is the local oncotic pressure difference across the endothelial surface glycocalyx and not the global difference between the plasma and tissue. In single frog mesenteric microvessels perfused and superfused with solutions containing 50 mg/ml albumin, the effective oncotic pressure exerted across the microvessel wall was not significantly different from that measured when the perfusate alone contained albumin at 50 mg/ml. Measurements were made during transient and steady-state filtration at capillary pressures between 10 and 35 cmH(2)O. A cellular-level model of coupled water and solute flows in the interendothelial cleft showed water flux through small breaks in the junctional strand limited back diffusion of albumin into the protected space on the tissue side of the glycocalyx. Thus oncotic forces opposing filtration are larger than those estimated from blood-to-tissue protein concentration differences, and transcapillary fluid flux is smaller than estimated from global differences in oncotic and hydrostatic pressures.     

Structural basis for some permeability properties of the air--blood barrier.

The structural basis for the permeability of the alveolar-capillary membrane to water-soluble solutes rests in part on the structure and function of its intercellular junctions and the pinocytotic vesicles within its cells. Intercellular junctions between endothelial cells of the pulmonary capillary bed differ both in permeability to enzyme tracers and in their structure. As determined by freeze fracture, the junctions in the arteriolar, capillary, and venular portion of the capillary network vary in complexity, and in the number of rows of particles constituting the junction. Because there are few particles associated with the junctions in the venular end of the capillary bed, these are considered to be the most permeable of the three types of vascular junctions. Epithelial junctions, in contrast, are impermeable to all enzyme tracers studied, and they are composed of a continuous, complex network of junctional fibrils. While intercellular junctions form seals of varying 'tightness,' pinocytotic vesicles provide a means for the transport of water-soluble macromolecules across the alveolar-capillary membrane.     

Explaining glomerular pores with fiber matrices. A visualization study based on computer modeling.

The extracellular space of the glomerular capillary wall is occupied by a complex meshwork of fibrous molecules. Little is understood about how the size, shape, and charge recognition properties of glomerular ultrafiltration arise from this space-filling fiber matrix. We studied the problem of size recognition by visualizing the void volume accessible to hard spheres in computer-generated three-dimensional homogeneous random fiber matrices. The spatial organization of the void volume followed a complex "blob-and-throat" pattern in which circumscribed cavities of free space within the matrix ("blobs") were joined to adjacent cavities by narrower throats of void space. For sufficiently small solutes, chains of blobs and throats traversed the matrix, providing pathways for trans-matrix permeation. The matrices showed threshold or gating properties with respect to permeation: solutes whose radius exceeded a critical value, at which a throat on the last connected trans-matrix pathway pinched off, could not cross, whereas smaller solutes had nonzero permeability. The thresholds may give the glomerular fiber matrix porelike response properties and explain why pore models have been such a useful means of treating permselectivity.     

Transport of ions across the blood-brain barrier

Capillaries in the brain are formed by a uniquely specialized endothelial cell that regulates the movement of substances between blood and brain. Although they provide an impermeable barrier to some solutes, brain capillary endothelial cells facilitate the transcapillary exchange of others. In addition, they contain specific enzymes that contribute to a metabolic blood-brain barrier by limiting the movement of compounds such as neurotransmitters across the capillary wall. Studies of sodium and potassium transport by brain capillaries indicate that the endothelial cell contains distinct types of ion transport systems on the two sides of the capillary wall, i.e., the luminal and antiluminal membranes of the endothelial cell. As a result, specific solutes can be pumped across the capillary against an electrochemical gradient. These transport systems are likely to play a role in the active secretion of fluid from blood to brain and in maintaining a constant concentration of ions in the brain's interstitial fluid. In this way, the brain capillary endothelium is structurally and functionally related to an epithelium.     

Apparent Diffusivity and Taylor Dispersion of Water and Solutes in Capillary Beds

A physical theory explaining the anisotropic dispersion of water and solutes in biological tissues is introduced based on the phenomena of Taylor dispersion, in which highly diffusive solutes cycle between flowing and stagnant regions in the tissue, enhancing dispersion in the direction of microvascular flow. An effective diffusion equation is derived, for which the coefficient of dispersion in the axial direction (direction of capillary orientation) depends on the molecular diffusion coefficient, tissue perfusion, and vessel density. This analysis provides a homogenization that represents three-dimensional transport in capillary beds as an effectively one-dimensional phenomenon. The derived dispersion equation may be used to simulate the transport of solutes in tissues, such as in pharmacokinetic modeling. In addition, the analysis provides a physically based hypothesis for explaining dispersion anisotropy observed in diffusion-weighted imaging (DWI) and diffusion-tensor magnetic resonance imaging (DTMRI) and suggests the means of obtaining quantitative functional information on capillary vessel density from measurements of dispersion coefficients. It is shown that a failure to account for flow-mediated dispersion in vascular tissues may lead to misinterpretations of imaging data and significant overestimates of directional bias in molecular diffusivity in biological tissues. Measurement of the ratio of axial to transverse diffusivity may be combined with an independent measurement of perfusion to provide an estimate of capillary vessel density in the tissue.     

Colorimetric assay to quantify macromolecule diffusion across endothelial monolayers

Endothelial "capillary leak", the loss of vascular integrity in response to noxious stimuli, is characterized by extravasation of protein-richfluidfrom capillary lumen into surrounding tissue interstitium. This increase in vascular permeability, in response to inflammatory mediators, correlates with endothelial cell contraction and the formation of intercellular gaps within the monolayer. However, in vivo assessment of paracellular solute flow between endothelial cells may be complicated by multiple uncontrolled parameters. In vitro examinations of endothelial barrier leak have relied on electrical impedence or macromolecule diffusion techniques to determine the details pertinent to capillary barrier function. In this report, a simple, sensitive, nonradioactive, colorimetric assay to quantify the leak of a labeled protein marker across endothelial monolayers is described. This procedure avoids the hazards of radioisotope labels and the technical limitations of electrical resistance technology.     

Increased expression of β-catenin in brain microvessels of a segmentally trisomic (Ts65Dn) mouse model of Down syndrome

We examined the distribution of β-catenin and endogenous blood serum albumin at the ultrastructural level in blood microvessels (capillaries) from brains of control and trisomic Ts65Dn mice. Morphological examination revealed an increased immunolabeling for β-catenin in endothelial substructures of the capillary network, such as intercellular junctions, cytoplasm, and nuclei. These immunosignals were significantly increased in all endothelial substructures from trisomic mice. These changes, however, did not affect the blood–brain barrier function of the entire microvascular network, because the increased uptake of albumin by endothelial cells and the eventual escape of this protein (microleakage) into the perivascular neuropil were noted only in a few capillary profiles. Nevertheless, these findings suggest the involvement of some segments of the microvascular network in the brain pathology associated with DS.     

Transient transcapillary exchange of water driven by osmotic forces in the heart

Osmotic transient responses in organ weight after changes in perfusate osmolarity have implied steric hindrance to small-molecule transcapillary exchange, but tracer methods do not. We obtained osmotic weight transient data in isolated, Ringer-perfused rabbit hearts with NaCl, urea, glucose, sucrose, raffinose, inulin, and albumin and analyzed the data with a new anatomically and physicochemically based model accounting for 1) transendothelial water flux, 2) two sizes of porous passages across the capillary wall, 3) axial intracapillary concentration gradients, and 4) water fluxes between myocytes and interstitium. During steady-state conditions approximately 28% of the transcapillary water flux going to form lymph was through the endothelial cell membranes [capillary hydraulic conductivity (Lp) = 1.8 +/- 0.6 x 10-8 cm. s-1. mmHg-1], presumably mainly through aquaporin channels. The interendothelial clefts (with Lp = 4.4 +/- 1.3 x 10-8 cm. s-1. mmHg-1) account for 67% of the water flux; clefts are so wide (equivalent pore radius was 7 +/- 0.2 nm, covering approximately 0.02% of the capillary surface area) that there is no apparent hindrance for molecules as large as raffinose. Infrequent large pores account for the remaining 5% of the flux. During osmotic transients due to 30 mM increases in concentrations of small solutes, the transendothelial water flux was in the opposite direction and almost 800 times as large and was entirely transendothelial because no solute gradient forms across the pores. During albumin transients, gradients persisted for long times because albumin does not permeate small pores; the water fluxes per milliosmolar osmolarity change were 200 times larger than steady-state water flux. The analysis completely reconciles data from osmotic transient, tracer dilution, and lymph sampling techniques.     

Thrombospondin-1 induces tyrosine phosphorylation of adherens junction proteins and regulates an endothelial paracellular pathway

Thrombospondin-1 (TSP) induces endothelial cell (EC) actin reorganization and focal adhesion disassembly and influences multiple EC functions. To determine whether TSP might regulate EC-EC interactions, we studied the effect of exogenous TSP on the movement of albumin across postconfluent EC monolayers. TSP increased transendothelial albumin flux in a dose-dependent manner at concentrations >/=1 microg/ml (2.2 nM). Increases in albumin flux were observed as early as 1 h after exposure to 30 microg/ml (71 nM) TSP. Inhibition of tyrosine kinases with herbimycin A or genistein protected against the TSP-induced barrier dysfunction by >80% and >50%, respectively. TSP-exposed monolayers exhibited actin reorganization and intercellular gap formation, whereas pretreatment with herbimycin A protected against this effect. Increased staining of phosphotyrosine-containing proteins was observed in plaque-like structures and at the intercellular boundaries of TSP-treated cells. In the presence of protein tyrosine phosphatase inhibition, TSP induced dose- and time-dependent increments in levels of phosphotyrosine-containing proteins; these TSP dose and time requirements were compatible with those defined for EC barrier dysfunction. Phosphoproteins that were identified include the adherens junction proteins focal adhesion kinase, paxillin, gamma-catenin, and p120(Cas). These combined data indicate that TSP can modulate endothelial barrier function, in part, through tyrosine phosphorylation of EC proteins.     

An osmotic hypothesis for the regulation of oxygen permeability in soybean nodules

Nodule permeability (P) controls the amount of O₂ entering the nodule, and thereby the rates of both nodule respiration and N₂ fixation. P may be regulated by changes in the effective thickness of a water-filled diffusion barrier in the nodule cortex. Regulation of diffusion barrier thickness was hypothesized to result from changes in the water content of intercellular spaces. Modulation of intercellular water would be a response to osmotic potential gradients in the tissue. To test this hypothesis, preliminary experiments examined three classes of solutes (soluble sugars, free amino acids, and ureides) in nodules of intact plants exposed to 10 or 21 kPa O₂ for 24 h. Neither soluble sugars nor free amino acids in nodules were responsive to O₂ treatments. However, nodule ureides accumulated after exposure to 10kPa O₂ for 24 h. A symplastic increase in nodule ureides under the 10kPa O₂ treatment compared to the 21 kPa O₂ treatment may have removed water from intercellular spaces in the nodule cortex and increased P. In addition, the nodule cortex of intact plants was infiltrated with water, polyethylene glycol (PEG), KC1, or Na-succinate solutions to determine the effect of intercellular water and osmoticants on dinitrogenase activity and P. Results from infiltrating the apoplast of the nodule cortex with osmotic solutions indicated that both increases in intercellular water and decreases in the apoplastic water potential decrease dinitrogenase activity and P. Furthermore, the inability to recover dinitrogenase activity and P following the infiltration of the cortex with PEG compared to either KCl or Na-succinate treatments may indicate that recovery was dependent upon removal of the solute from the apoplast.     

A steady-state filtration model for transluminal water movement in small and large blood vessels

It is now generally accepted that the intercellular cleft between adjacent endothelial cells is the primary pathway for the transluminal movement of water and small ions in the vasculature. A steady-state theoretical model has been developed to show quantitatively how the geometry of the intercellular cleft between adjacent endothelial cells is related to both the water movement and pressure distribution in the subendothelial space and to examine how the existence of a subendothelial interaction layer affects the hydraulic resistance of the media of vessels of varying wall thickness. The velocity and pressure fields in the media are described using porous matrix theory based on Darcy's law and a lubrication-type analysis is used to describe the flow in a variable geometry intercellular cleft. These two equations are solved simultaneously to determine the unknown pressure distribution beneath the endothelium and the flow in the arterial media. Application of this model shows that, when the tight junction in the cleft is 26 A or less, more than half of the total hydraulic resistance of the wall occurs across the endothelial cell monolayer, for a vessel whose wall thickness is less than 0.02 cm. This finding is in good agreement with the experimental findings of Vargas, et al. (1978) for rabbit aorta. Contrary to previous belief, the model shows that the filtration resistance of an arterial wall with intact endothelium does not scale linearly with wall thickness due to the highly nonlinear resistance of the endothelial interaction layer.     

Growth of the plant cell wall

Plant cells encase themselves within a complex polysaccharide wall, which constitutes the raw material that is used to manufacture textiles, paper, lumber, films, thickeners and other products. The plant cell wall is also the primary source of cellulose, the most abundant and useful biopolymer on the Earth. The cell wall not only strengthens the plant body, but also has key roles in plant growth, cell differentiation, intercellular communication, water movement and defence. Recent discoveries have uncovered how plant cells synthesize wall polysaccharides, assemble them into a strong fibrous network and regulate wall expansion during cell growth.     

Selective Filtering of Particles by the Extracellular Matrix: An Electrostatic Bandpass

The transport of microscopic particles such as growth factors, proteins, or drugs through the extracellular matrix (ECM) is based on diffusion, a ubiquitous mechanism in nature. The ECM shapes the local distribution of the transported macromolecules and at the same time constitutes an important barrier toward infectious agents. To fulfill these competing tasks, the hydrogels have to employ highly selective filtering mechanisms. Yet, the underlying microscopic principles are still an enigma in cell biology and drug delivery. Here, we show that the extracellular matrix presents an effective electrostatic bandpass, suppressing the diffusive motion of both positively and negatively charged objects. This mechanism allows uncharged particles to easily diffuse through the matrix, while charged particles are effectively trapped. However, by tuning the strength of this physical interaction of the particles with the biopolymer matrix, the microscopic mobility of formerly trapped particles can be rescued on demand. Moreover, we identify heparan sulfate chains to be one important key factor for the barrier function of the extracellular matrix. We propose that localized charge patches in the ECM are responsible for its highly unspecific but strongly selective filtering effect. Such localized interactions could also account for the observed tunability and selectivity of many other important permeability barriers that are established by biopolymer-based hydrogels, e.g., the mucus layer of endothelial cells or the hydrogel in the nuclear core complex.     

Quantitative immunocytochemical studies of endogenous albumin in rat aortic endothelial and mesothelial cells

Endogenous albumin was revealed over cellular structures of rat ascendent aorta endothelia and mesothelium, with high resolution and specificity, by applying the protein A-gold immunocytochemical approach. This approach allows albumin distribution to be studied under steady-state conditions. The cellular layers evaluated were the aortic endothelium, the capillary endothelium (vasa vasorum), and the mesothelium externally lining the aorta at this level. Gold particles, revealing albumin antigenic sites, were preferentially located over plasmalemmal vesicles and intercellular clefts of endothelial and mesothelial cells, though with different labeling intensities. The interstitial space was also labeled. Morphometrical evaluation of plasmalemmal vesicles demonstrated a higher surface density for these structures in capillary endothelial cells (12%) compared with those in aortic endothelial (5%) and mesothelial cells (2%). Quantitation of gold labeling intensities over these structures revealed a higher labeling over plasmalemmal vesicles of capillary endothelium than over those of aortic endothelium and mesothelium. This result, together with the higher surface density of plasmalemmal vesicles found in capillary endothelium, suggest an important role of these structures in the transendothelial passage of endogenous albumin, particularly for capillary endothelium. On the other hand, labeling densities over mesothelial clefts were found to be higher than those of capillary and aortic endothelia. Results from this study concur with the proposal of a differential passage of albumin according to the cell lining considered, and suggest to a role for mesothelial intercellular clefts in contributing to the presence of albumin in interstitial spaces.     

Fibrinogen induces endothelial cell permeability

Many cardiovascular and cerebrovascular disorders are accompanied by an increased blood content of fibrinogen (Fg), a high molecular weight plasma adhesion protein. Fg is a biomarker of inflammation and its degradation products have been associated with microvascular leakage. We tested the hypothesis that at pathologically high levels, Fg increases endothelial cell (EC) permeability through extracellular signal regulated kinase (ERK) signaling and by inducing F-actin formation. In cultured ECs, Fg binding to intercellular adhesion molecule-1 and to α₅β₁ integrin, caused phosphorylation of ERK. Subsequently, F-actin formation increased and coincided with formation of gaps between ECs, which corresponded with increased permeability of ECs to albumin. Our data suggest that formation of F-actin and gaps may be the mechanism for increased albumin leakage through the EC monolayer. The present study indicates that elevated un-degraded Fg may be a factor causing microvascular permeability that typically accompanies cardiovascular and cerebrovascular disorders.     

Localization and distribution of anionic charges in the glomerular mesangium of normal and nephrotic rats

Sulfated glycosaminoglycans and sialoglycoproteins are thought to play a pivotal role in the glomerular capillary wall barrier to filtration since these anionic charged elements are important in the maintenance of capillary wall integrity and constitute a charge-selective filter. The development of proteinuria in puromycin aminonucleoside (PAN) nephrosis is associated with polyanion loss from the glomerular capillary wall structures. Since in PAN nephrosis the permeability of the mesangial area to plasma proteins and tracer substances has also been shown to be increased, the purpose of this study was to analyse the localization and distribution of anionic charges in the glomerular mesangium in this experimental model. Glycosaminoglycans were labeled by perfusion of the kidneys with ruthenium red solution (RR). Electron microscopic examination revealed the presence of distinct small RR granules ("anionic sites") in the mesangial intercellular matrix substance and in the laminae rarae of the glomerular basement membrane (GBM). The center-to-center spacing of the granules was measured and a frequency distribution of intervals in different interval classes was constructed. In normal glomeruli the anionic sites in the mesangial matrix showed a distribution pattern identical to the GBM with a maximal interval incidence at the 31-40 nm class. In nephrotic rats anionic site distributions in matrix and GBM did not change significantly. Sialoglycoproteins were labeled with colloidal iron (CI). In PAN nephrosis a decrease of CI binding was observed at the epithelial-basement membrane junction of the glomerular capillary wall. However, CI labeling of the mesangial matrix and mesangial cell membranes did not differ from that of normal glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)