Microvascular Permeability and the Exchange of Water and Solutes Across Microvascular Walls

Exchange through microvascular walls is both the initial and the final step of transport of materials by the circulation. In most tissues, microvascular exchange is a passive process, driven by differences in hydrostatic pressure and solute concentration between the circulating plasma and the interstitial fluid that flank microvessel walls. Lipophilic molecules and small water-soluble molecules and ions can exchange rapidly in most vascular beds, but microvascular walls are a barrier to macromolecules, severely impeding their exchange. The consequent differences in macromolecular concentration across microvascular walls are responsible for differences in osmotic pressure, which were identified over a century ago to play an essential role in the balance of fluid between the circulating blood and the tissues.

Introduction

Although small hydrophilic molecules can exchange rapidly between the blood and the tissues, it is wrong to assume that microvascular walls are no barrier to them. The rate at which changes of concentration of a solute in the blood can be reflected as changes in interstitial fluid (ISF) concentration depends both on the blood flow to the capillary beds, and on the permeability of the capillary walls to the solute. It is often assumed that for small molecules only blood flow is important, but once flow has exceeded a certain minimum range of values, the rate of equilibration of blood and ISF becomes limited by permeability.

Microvascular exchange and microvascular permeability are sometimes regarded as synonymous but this in incorrect and it is important to distinguish between them. Microvascular permeability to a substance is the conductivity of the microvessel wall and the permeability coefficients which describe permeability in functional terms are determined by the structure and properties of the pathways through which its molecules traverse the vessel wall. Microvascular exchange rates and net transport through microvascular walls for a particular substance are determined by microvascular permeability to that substance and also by the value and direction of net fluid flow through the vessel wall and by differences in concentration of the substance between plasma and ISF. The difference between microvascular exchange and microvascular permeability is important when interpreting methods that claim to demonstrate changes in permeability. Many of these methods involve the measurement of changes in net transport of macromolecules between blood and tissues, without controlling the transcapillary differences in hydrostatic pressure and macromolecular concentration. We shall consider the potential errors later in this chapter.

First, we consider microvascular permeability, and then microvascular exchange. Not included in this chapter is a consideration of transport through the walls of microvessels of the central nervous system. The low permeabilities of these vessels and the specialized transport mechanisms for certain solutes constituting the blood–brain barrier are atypical of other microvessels, and resemble those of tight epithelia.

Microvascular Permeability

Microvascular Ultrastructure

Capillary walls consist of a single layer of flattened endothelial cells, the endothelia, and these cells constitute the barrier between the blood and the ISF. Electron microscopy has revealed that endothelial cells in different tissues are of two distinct types: “continuous” and “fenestrated” ( Figure 9.1 ). Continuous endothelium is found in microvessels of skin, muscle, lung, and connective tissues. Here, the endothelial cells are joined together by tight junctions to form a continuous layer surrounded by a continuous basement membrane. The plasmalemmal membranes of the continuous endothelia retain their integrity; even in areas where the cells are flattened, reducing their thickness to less than 0.1 μm, the distinct luminal and abluminal membranes are separated by a thin layer of cytoplasm.

Figure 9.1, Diagrams showing the ultrastuctural features of microvascular walls in transverse section: (a) Vessel with continuous endothelium; (b) Vessel with fenestrated endothelium.

Fenestrated endothelium is found in microvessels associated with secretory and absorptive epithelia, e.g., the capillaries of the intestinal mucosae, glomerular, and peri-tubular capillaries of the kidney. The walls of fenestrated microvessels are also made of a single continuous layer of endothelial cells joined by tight junctions and surrounded by a continuous basement membrane, but in these vessels attenuated areas of cells appear to be penetrated by circular openings 40 to 70 nm in diameter. These are the fenestrae (or fenestrations), and in most cases the fenestrations are closed by a thin electron-dense diaphragm, which appears to be arranged as a series of broad spokes with central “hub” ( Figure 9.2 ).

Figure 9.2, En face view of fenestrated endothelium of peritubular capillary of the kidney in a rapid freeze deep etch preparation.

Covering the luminal surface of endothelial cells is a layer of glycoprotein called the glycocalyx or endocapillary layer (ECL). First identified by Luft in 1966 using ruthenium red staining, its importance has only come to be widely appreciated in the past 15 years. Although both continuous and fenestrated endothelia have been found to contain the various inclusions common to most cells (e.g., mitochondria, rough and smooth endoplasmic reticulum) the dominating ultrastructural feature seen in transmission electron micrographs is the large number of small endoplasmic vesicles ( Figure 9.3 ). The majority of vesicles are arranged in fused clusters that communicate with each other and with flask like pits on either the luminal or abluminal surfaces of the cells called the caveolae intracellulares. Chains of fused vesicles forming channels that pass through endothelial cells appear to be relatively rare occurrences in unstimulated endothelium, but are a feature of endothelium activated by certain mediators.

Figure 9.3, Electron micrograph of microvascular endothelium of frog.

The intercellular clefts of continuous endothelia, the fenestrae of fenestrated endothelia, and the small vesicles have all been implicated as pathways through the endothelia as a result of experiments using electron-dense tracers. Controversy surrounded the interpretation of many of these experiments, but progress has been made over the past 20 years as physiological evidence clarified their interpretation. For this reason, the ultrastructural basis of permeability is considered after we have defined the permeability coefficients by considering the principles of passive transport and have discussed their values in different endothelia to different types of molecules.

Passive Transport and Permeability Coefficients of Porous Membranes

The mechanisms of transport are convection and diffusion. Convection is equivalent to bulk flow of solutions and the solutes within them. Thus, if a solution flows from a reservoir A at a high pressure to a second reservoir, B, at a lower pressure, there is bulk flow of the solution and transport of the solutes by convection. If J _V ml sec ⁻¹ is the rate at which the solution flows from A to B, it is proportional to the difference in hydrostatic pressure, ΔP , between A and B as follows:

J_{V} = K Δ P

$J_{V} = K Δ P$

where K is the conductance of the system between A and B.

If A and B are separated by a porous membrane, K is proportional to the area of the membrane, A _m , through which fluid can flow. Thus:

J_{V} = L_{P} A_{m} Δ P

$J_{V} = L_{P} A_{m} Δ P$

and

L_{P} = (J_{V} / A_{m} Δ P)

$L_{P} = (J_{V} / A_{m} Δ P)$

where L _P is the hydraulic conductivity or hydraulic permeability of the membrane. L _P is one of several membrane permeability coefficients. It describes the ease with which fluid flows through a unit area of membrane when driven by a unit difference in pressure. It can be thought of as describing the frictional interactions between the membrane molecules and the molecules of the solution (principally water), and like the other permeability coefficients its value depends on the structure of the membrane. If small water-filled pores penetrate the membrane, L _P is proportional to the number of pores per unit area of membrane, to the fluid viscosity, and to a function of the dimensions of the pores. This function depends on the size and shape of the pores and the nature of flow through them.

Equation (9.2) describes the flow of a solution through a membrane only if the solute molecules can pass through the membrane as easily as the water molecules. If movement of solute is hindered to a greater extent than the water, filtration of the solution leads to the development of a solute concentration difference across the membrane as water enters compartment B faster than solute. The difference in solute concentration gives rise to a difference in osmotic pressure which opposes filtration of the solution across the membrane. The force driving solution through the membrane is no longer just ΔP , but ΔP minus the effective osmotic pressure between the solutions in A and B. The magnitude of this osmotic pressure difference is related not only to the concentration difference itself, but also to the degree to which the membrane hinders the movement of solute relative to water. This is described by a second membrane coefficient, σ _d , referred to as the osmotic reflection coefficient. If the channels in the membrane are permeable to water but not solute molecules, filtration of a solution of this solute through the membrane will separate the solute from the water, with pure water leaving the membrane and concentration of the solution upstream. Since all the solute molecules are reflected at the membrane, σ _d has a value of 1.0. The osmotic pressure exerted by differences in its concentration across this particular membrane should be close to that calculated from Van’t Hoff’s law, i.e., Δ Π = RTΔC , depending on the “ideality” of the solution. If, alternatively the solute molecules are able to pass through the membrane as easily as the water molecules, σ _d has a value of 0, as none of the solute molecules are reflected. If the solute passes the membrane more easily than water, σ _d has a negative value. If only a fraction of the solute molecules are “reflected” during ultrafiltration of the solution, σ _d has a value between zero and one.

Only those molecules that are reflected at the membrane during ultrafiltration can exert an osmotic pressure across it. Thus, the osmotic pressure difference, ΔΠ, is related to the concentration difference, Δ C , through the universal gas constant, R , and the absolute temperature, T :

Δ Π = σ_{d} R T Δ C

$Δ Π = σ_{d} R T Δ C$

and

σ_{d} = \frac{Δ Π}{R T Δ C}

$σ_{d} = \frac{Δ Π}{R T Δ C}$

At microvascular walls, macromolecules such as plasma proteins have high values of σ _d and σ _f (0.8–0.999), whereas small ions (e.g., Na ⁺ , K ⁺ , Cl ⁻ ) and small hydrophilic molecules (e.g., glucose, lactic acid, amino acids, urea) have values of σ less than 0.2 in most vessels.

Net fluid flow through microvascular walls carries dissolved solutes by convection. The rate of solute transport by convection from A to B depends on J _V , and also upon the value of σ for the particular solute at the membrane. If σ has a value of zero, then J _S is equal to J _V C (in moles per second), where C is the solute concentration in the solution (in moles per ml), i.e., the solution flowing out of the membrane into B has the same concentration as that entering the membrane from A. If, however, σ has a value between zero and one, then the solution emerging from the membrane into B will have a lower solute concentration than that entering the membrane at A. Convective transport of solute through the membrane is now described by the relation:

J_{S} = J_{V} (1 - σ_{f}) C

$J_{S} = J_{V} (1 - σ_{f}) C$

where C now refers to the concentration entering the membrane from A. Note that in Eq. (9.4) σ is written as σ _f , whereas in describing the effective osmotic pressure σ is written as σ _d . For “an ideal solute” σ _f = σ _d , but solutions of macromolecules deviate from “ideal” behavior and σ _d is often measurably greater than σ _f . Rearrangement of Eq. (9.3) provides a definition of σ _f :

σ_{f} = 1 - \frac{J_{S}}{J_{V} C}

$σ_{f} = 1 - \frac{J_{S}}{J_{V} C}$

If the only pathway for both water and solutes through the membrane is a population of equally sized channels, σ _d and σ _f are determined only by the ratio of the dimensions of the solute molecules to those of the channels, and are independent of the number of channels per unit area of membrane.

A second mechanism of transport through porous membranes is diffusion. This is most important for the transport of small molecules across microvascular walls. In contrast to the stately progression of molecules by convection, diffusion results from the random jostling of all molecules in a solution that represents their thermal energy. Diffusion is a mixing process, and where there are differences in concentration in a solution diffusion is responsible for the spontaneous net transport of solute from regions of high to regions of low concentration. On a macroscopic scale, Fick’s first law of diffusion describes net transport of solute by diffusion:

J_{S} = D A (\frac{- d C}{d x})

$J_{S} = D A (\frac{- d C}{d x})$

where D is the diffusion coefficient of the solute in the solution, A is the area through which diffusion occurs, and the derivative (− dC/dx ) is the concentration gradient of solute down which diffusion occurs. The negative sign of the derivative is to indicate that diffusion occurs from a region of high concentration (low value of x) to a region of lower concentration (higher value of x), i.e., the diffusion of a solute is directed down its concentration gradient.

Diffusion coefficients of solutes in aqueous solutions reflect the ability of solute molecules to slip past adjacent molecules of water. They are measured and defined in terms of net movements of solute under conditions in which there is no overall movement of the solution. Thus, diffusion in solution is a displacement process whereby the displacement of a solute molecule in one direction is accompanied by the displacement of an equal volume of water in the opposite direction. Because the rate at which displacement occurs is dependent on the thermal energy of the solution, the diffusion coefficient is directly proportional to temperature. It is also inversely proportional to the frictional interactions between the solute and water molecules.

When diffusion of a solute occurs through a thin porous membrane, the diffusional permeability of the membrane to the solute, P _d , is defined in terms of the net solute flux, J _S , the solute concentration difference across the membrane, ΔC , and the membrane area, A _m , under conditions where there is no volume flow through the membrane. Thus:

P_{d} = {(\frac{J_{S}}{A_{m} Δ C})}_{J_{V} = 0}

$P_{d} = {(\frac{J_{S}}{A_{m} Δ C})}_{J_{V} = 0}$

P _d has units of velocity (cm sec ⁻¹ ), and like L _P and σ _f , it has meaning in terms of the interactions between the solute molecules, the molecules of the membrane, and the water within the membrane. Such interactions depend critically upon the ultrastructure of the pathways for solute and water through the membrane, particularly when the width of the pathways is comparable to the diameter of the diffusing molecules.

Diffusional permeability coefficients are defined under conditions where there is no net volume flow through the membrane. In measuring P _d of microvascular walls, it is often convenient to use radioactive isotopes or fluorescent tracers, because their fluxes can be detected when their concentration differences are in the micromolar range. Larger differences in concentration may set up significant differences in osmotic pressure, which may complicate the estimation of P _d by giving rise to net fluid flows in the opposite direction to those of the diffusing molecules.

Permeability to Lipophilic Solutes

Solutes that can dissolve to a significant extent in lipids have high cell membrane permeabilities, and very high microvascular permeabilities. It is assumed that these molecules diffuse directly through the entire microvascular wall. This group of solutes includes gas molecules, including O ₂ and CO ₂ , as well as N ₂ , the inert gases, and molecules of general anesthetics, although recent evidence suggests CO ₂ and NH ₃ may cross membranes additionally through specialized channels.

So rapidly can small lipophilic solutes cross microvascular walls that, under physiological conditions, their transport between the blood and the tissues is always limited by their rate of delivery or clearance by blood flow through the microcirculation, and it has been impossible to estimate their microvascular permeability coefficients with accuracy with values for some greater than 10 ⁻² cm.sec ⁻¹ .

Microvascular permeability to lipophilic molecules is, however, sensitive to temperature. Renkin showed that antipyrene, which is fat-soluble at body temperature, could exert substantial osmotic pressures across the walls of capillaries in cat hind limbs when the tissue was cooled to 15°C, but not when the tissue was at 37°C. A similar phenomenon has been demonstrated by Curry in single mesenteric capillaries.

Permeability Coefficients to Small Hydrophilic Molecules

Microvascular permeability to macromolecules is usually considered separately from permeability to hydrophilic molecules smaller than serum albumin, as different mechanisms of transport appear to be involved. Evidence for this emerged in 1956 from studies on the passage of a range of dextran polymers between plasma and lymph. Some of the original data are shown in Figure 9.4 , where it is seen that the steady-state concentrations of the smaller dextrans in the lymph draining hind limb tissues of anesthetized dogs relative to the plasma levels fall rapidly as their molecular radii increase up to 4–5 nm; for the larger molecules the decline of lymph concentration with molecular size is so small as to appear barely significant in these data.

Figure 9.4, Relations between permeability (P) of dog hind limb microvessels to hydrophilic solutes and the molecular diameter of the solutes.

This two-component relation between P _d and molecular diameter is seen in nearly all microvascular beds, suggesting that small molecules use different mechanisms or pathways to cross microvascular walls from those used by macromolecules.

Permeability Coefficients to Small Hydrophilic Molecules

In Figure 9.5 , the values of P _d measured in microvascular beds of skeletal muscle and single mesenteric microvessels to some small water soluble solutes have been plotted against solute molecular radius. In mesenteric capillaries, values for P _d to the smallest molecules and ions (e.g., Na ⁺ , K ⁺ , urea) are ten times greater than values of P _d for the same solutes in muscle capillaries. To make the comparison easier, both the P _d values and values for molecular radius are shown on logarithmic scales. The decline of P _d with molecular radius for both types of microvessel is much greater than the decline of diffusion coefficient ( D ) for the same molecules in open aqueous solution. The more rapid decline of P _d can be accounted for if molecules were diffusing through water-filled channels whose widths were comparable to their own diameters. The smooth curves, which have been fitted to the data, converge as molecular radius increases. If the channels were cylindrical pores, the convergence implies that the radii of the pores is very similar in mesenteric and muscle capillaries, but there are ten times more pores penetrating per unit area of the walls of mesenteric capillaries than of muscle capillaries. The value of molecular radius at which the curves intersect provides an estimate of the pore radius – in this case between 3.5 and 4 nm.

Figure 9.5, Relations between the logarithms of P d and molecular radius for small to intermediate sized hydrophilic solutes in capillaries of frog mesentery (filled circles) and cat skeletal muscle (filled triangles).

This pattern of declining P _d to small hydrophilic solutes with increasing molecular diameter is seen in all microvessels where it has been sought, and curves based on the theory of diffusion through pores of radii between 3.5 nm–5 nm can be fitted to these data.

The values of P _d to a particular small hydrophilic solute (e.g., Na ⁺ , K ⁺ , sucrose, and inulin) in different microvascular beds correlate with the values of hydraulic permeability ( L _P ). This is shown in Figure 9.6 , where each point represents the mean value of L _P plotted against the mean value of P _d to Na ⁺ or K ⁺ in the same microvessel or microvascular bed, different points representing values for vessels in different tissues. The data have been plotted on logarithmic scales so that values covering two orders of magnitude can be compared. The slope that has been drawn through the points has been given a value of unity, to indicate that direct proportionality is a reasonable description of the relationship. The correlation is strong circumstantial evidence for believing that the same pathways through the endothelium serve both for the rapid exchange of small hydrophilic solutes, and for most net fluid movements. Furthermore, because Na ⁺ ions are likely to follow an extracellular route, it seems likely that this shared pathway is extracellular. This line of reasoning is greatly strengthened by the demonstration of similar linear correlations between L _P and P _d values for other extracellular solutes, such as sucrose and inulin in different microvascular beds.

Figure 9.6, Variations in L P and P d to small ions (Na + and K + ) in different microvascular beds.

Although the exchange of small hydrophilic molecules occurs through the same channels that account for most of L _{P ,} some water may also cross microvascular walls of continuous endothelium by channels not available to solutes. This additional route has been identified by Pallone et al. as channels formed by the membrane protein aquaporin-1, AQP-1. The first real evidence for a “water only” pathway was indicated by Yudilevich and Alvarez from measurements of the rates of diffusion of triated water and Na ⁺ through dog heart capillaries. From measurements of σ to small hydrophilic solutes, Curry and colleagues estimated that in single perfused capillaries between 5 and 10% of the pathways responsible for L _P were available only to water, and not to small solutes. More recently, Turner and Pallone have shown that “water only” channels account for a similar proportion of the L _P of descending vasa recta of rat kidney. It is worth emphasizing that although fluid movements through AQP-1 channels of the descending vasa recta are of physiological importance, the channels contribute no more than 5 to 10% to the total L _P of these vessels. Although it has been suggested that AQP-1 channels might contribute to as much as 30% of the L _P of skeletal muscle capillaries, the strong correlation between P _d and L _P shown in Figure 9.6 indicates that the contribution of AQP-1 channels to the L _P of most exchange vessels is small.

Although absolute values of L _P and P _d to small hydrophilic solutes vary greatly from one microvascular bed to another, the reflection coefficient to large molecules are remarkably similar in different vessels when the tissues are undamaged (or unstimulated). This is shown in Figure 9.7 , where the mean values of σ to serum albumin have been plotted against the mean value of L _P for various tissues. Whereas L _P varies over three orders of magnitude, σ to albumin is usually in the range of 0.8 to 1.0. There is no trend in the relation between σ _albumin and L _P . Vessels with high values of L _P have mean values of σ _albumin that are as high, if not higher, than in vessels with low values of L _P . This means that the channels or pores that are largely responsible for the L _P of vessels in different tissues all restrict the passage of albumin to a similar extent. Thus, both the variations of P _d with molecular size and the variations in σ _albumin and L _P in different capillary beds lead to the same conclusion: individual channels for exchange of water and small hydrophilic solutes are similar in different capillary types, but the density of these channels in vessel walls varies considerably.

Figure 9.7, Relation between the reflection coefficient to serum albumin (σ) and the L P in different microvascular beds.

Permeability Coefficients to Macromolecules

Apart from the sinusoids of the liver and spleen, microvascular walls have high reflection coefficients and low permeabilities to macromolecules. These permeability properties are essential if the circulating blood is to be retained within the vascular system. Although their permeabilities are low, macromolecules do cross the walls of all microvessels at slow but finite rates. Table 9.1 compares estimates of σ and P _d made from the transport of macromolecules between blood and lymph in the dog paw and the cat intestine. Although it is possible that the values given in this table are actually overestimates of the permeability coefficient of macromolecules, there are three important points to note here. First, the values of P _d for serum albumin are more than 3000 times less than the permeabilities of the same vessels to Na ⁺ and urea. The diffusion coefficient of albumin in aqueous solutions is only 20 times less than those of Na ⁺ and urea. This means that the diffusion of albumin molecules through microvascular walls is hindered 150 times more than the diffusion of Na ⁺ ions or urea molecules. Second, compared with the changes seen for small molecules, there is a relatively small decrease in P _d and rise in σ with increases in molecular size. The third point is the similarity of the values of P _d to the same macromolecule at the predominantly continuous endothelium of the dog paw capillaries, and also at the predominantly fenestrated endothelium of vessels in the small intestine. The values of L _P and P _d to small hydrophilic solutes in these two microvascular beds differ by nearly two orders of magnitude. This would seem to be further evidence supporting the belief that the transport pathways through microvascular walls are different for macromolecules from those responsible for water small hydrophilic solutes.

Table 9.1

Reflection Coefficients (σ) and Permeability Coefficients ( P _d ) to Macromolecules of Selected Molecular Radii (a _ES , nm) in Microvascular Beds of Dog Paw and Cat Ileum

			Dog Paw		Cat Ileum
Molecule	a _ES (nm)	σ	P _d ×10 ⁸ (cm sec ⁻¹ )	σ	P _d × 10 ⁸ (cm sec ⁻¹ )
Serum albumin	3.55	0.89	1.0–4.7	0.9	3.0
Transferrin	4.3	0.89	6.3	–	–
Haptoglobin	4.6	0.91	3.1	–	1.4
Immunoglobulin	5.6	0.91	3.3	0.95	–
Fibrinogen	10.0	0.94	1.6	0.98	0.7

Renkin, E. M. (1988). Transport pathways and processes. In “Endothelial Cell Biology,” 51–68, Simionescu, N., and Simionescu, M., (eds.) Plenum Publishing, New York.

It is possible that some of the values for P _d in Table 9.1 are overestimates and those for σ are underestimates because, in calculating vascular permeability coefficients from plasma to lymph transport, it is assumed that a steady-state has been established between the newly formed filtrate surrounding the microvessels and the ISF entering the lymph. Simple calculations suggest that in some tissues the time taken to reach a steady-state after a change in microvascular filtration rate can be many hours. This can be understood by considering the events that follow an increase in microvascular filtration. A rise in filtration rate is accompanied by a rise in lymph flow and a fall in the concentrations of macromolecules in the newly formed interstitial fluid. It may, however, take several hours before the lymph has the same composition as this new filtrate, and if the lymph is sampled prematurely its concentration of macromolecules will be greater than that in the ultrafiltrate. The lymph flow, however, may reach its new steady-state level before the lymph concentration of macromolecules, and if flux of macromolecules is estimated from values for the product of lymph flow and lymph concentration of macromolecules at this stage, its value will exceed the real rate of transport into the tissue. Not only is macromolecular transport overestimated, but the mean concentration differences across the microvascular walls are also underestimated.

Since 1990, much work has been conducted on the passage of macromolecules through monolayers of culture endothelial cells. In many of these studies, changes in permeability to macromolecules have been investigated, and relative rather than absolute values of permeability coefficients have been reported. In most but not all studies, absolute values for P _d of monolayers of cultured endothelial cells to albumin lie in the range of 10 ⁻⁶ cm sec ⁻¹ , about 100 times greater than the values of P _d to albumin at microvascular walls in situ . In a few laboratories, values in the range of 10 ⁻⁷ to 10 ⁻⁶ cm sec ⁻¹ have been reported, but even these values are at the very high end of the range found in microvessel walls in vivo . The reasons for this difference are not understood at present. Although studies on cultured endothelial cells have provided essential information of intracellular processes, conclusions from them relating to macromolecular permeability should be viewed with caution.

Like smaller hydrophilic molecules, the microvascular permeability coefficients to macromolecules decrease as molecular size increases, although the decline is considerably less steep. Molecular charge is also more important as molecular size increases. Evidence for the charge-selective nature of ultrafiltration in the renal glomerular capillaries is well-known, but in other microvessels, charge selectivity has been investigated in less detail. Areekul first provided evidence for charge selectivity in systemic capillaries. Working on the isolated perfused rabbit ear, he showed that σ _d to sulfated dextran (which is negatively charged) was always greater than σ _d to neutral dextran of the same molecular weight. Work on single perfused capillaries and on microvascular beds in rat hind limbs also supported the view of microvascular walls as a negatively-charged barrier.

This picture was temporarily confused by studies on the transport of charged macromolecules between plasma and lymph. Negatively-charged macromolecules appeared more rapidly in the lymph and at higher concentration than positively-charged molecules, suggesting a positively-charged barrier. It was then appreciated that the large number of fixed anionic sites in the interstitium (see ) would reduce the volume of distribution of the negatively-charged molecules relative to cationic or neutral molecules of the same molecular size. Parker and co-workers showed that the greater volume of distribution of neutral and cationic molecules increased the time for these molecules to reach a steady-state concentration in the ISF. This delay accounted for the apparently more rapid transport of the anionic molecules, a conclusion reinforced by the recent study of Gyenge and her co-workers on albumin distribution in skin and muscle tissue. With this point clarified, the evidence once again supports the concept of a negatively-charged barrier at microvascular walls hindering the transport of negatively-charged macromolecules greater than 3.0 nm in diameter.

The decline of P _d (and an increase in σ) with an increase in molecular size led to the suggestion that macromolecules cross the endothelium through a system of large pores with radii in the range of 20–40 nm. These pores would be few in number compared with the small pores that act as a pathway for small hydrophilic solutes. Calculations suggest that individual capillaries in skeletal muscle may have an average of only three large pores and with the usual variation between microvessels this indicates that many vessels will have no large pores at all. In a comprehensive review, Taylor and Granger showed that microvascular permeability to macromolecules in different tissues could be described by transport through a population of small pores (radii 3.5–5 nm) and a set of large pores (15–30 nm radii). If large pores do exist, then transport of macromolecules through them will be largely convective, sensitive to microvascular pressure, and increase with fluid filtration rate. Experiments by Rippe and his colleagues showed that this is the case for the transport of labelled albumin from capillaries in skeletal muscle of an isolated perfused rat limb preparation. Defining the clearance of tracer from the blood into the tissue as the rate of its accumulation divided by the perfusate concentration, they not only showed that clearance of albumin increased linearly with fluid filtration into the tissues, but also that cooling the tissue from 36° to 14°C reduced albumin clearance by only 40%. This was similar to the reduction of the apparent hydraulic permeability of the microvessels. The results of this classic experiment are shown in Figure 9.8 . A 40% reduction in L _P would be expected because the filtrate flow through the water-filled channels through the vessel walls is inversely proportional to fluid viscosity, and the viscosity of water at 36°C is approximately 60% of that at 14°C. Because albumin transported is proportional to filtration rate and is reduced in parallel to J _V as the viscosity of water is increased, Rippe and his colleagues concluded that albumin is transport through water-filled channels in the microvascular walls. Figure 9.8 shows that the clearance of albumin from plasma to tissue is significant when fluid filtration is zero. Rippe and his colleagues accounted for this by pointing out that, because the hydrostatic pressure difference across microvascular walls is greater than zero under these conditions, there would still be fluid filtration through the large pores, and consequent transport of albumin because σ _d to plasma proteins here is low. This filtration through the large pores would not be detected in whole tissue, because it would be balanced by an equal and opposite uptake of fluid through the “small pores” where σ _d to plasma proteins is high. As we shall see, the steady uptake of fluid from tissues into capillary blood may peter out and become a low level of filtration in tissues such as muscle.

Figure 9.8, The clearance of serum albumin from perfusate into tissues of rat skeletal muscle at different filtration rates in experiments conducted at 36°C and 13–15°C.

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