Helge Wiig, Christina C Gyenge, & Olav Tenstad
Division of Physiology, University of Bergen, Norway helge.wiig@biomed.uib.no

https://doi.org/10.36866/pn.56.29

The present article focuses on the interstitium, which is the physical and biochemical environment of cells. The interstitial space consists of connective and supporting tissues of the body and is located outside the blood, lymphatic vessels and parenchymal cells. A schematic of a generic interstitium is provided in Fig. 1A. Although the composition and structure of this physiological space varies from tissue to tissue, there are basic characteristics and functions that are representative of interstitia of most tissues. Essentially the interstitium can be divided into two phases: the interstitial fluid and the structural molecules of the interstitial or the extracellular matrix. As a generalized description, the interstitial extracellular matrix can be thought of as a three-dimensional ‘meshwork’ composed of a complex aggregation of protein fibres and carbohydrate polymers.

The presence of numerous interstitial macromolecules, particularly glycosaminoglycans (hyaluronan and proteoglycans) and collagen-based species, results in macromolecular crowding of the interstitial space. Consequently, the fluid space available for other species diffusing through the interstitial media is less than the total interstitial fluid volume, i.e. a given interstitial solute will distribute itself in the fluid space outside the meshwork or, alternatively, through those spaces of the meshwork that have dimensions larger than that of the solute. This phenomenon of geometrical, or sterical interstitial exclusion was first described by Ogston and Phelps ( 1961) and refers to the fact that two solid structures cannot occupy the same confined volume at the same time, as illustrated in Fig. 1B. The steric exclusion phenomenon is relevant only for species with high hydrodynamic sizes such as proteins and not for small molecules such as water, small ions and nutrients.

In addition to the steric exclusion, due to the fact that glycosaminoglycans are negatively charged at physiological pH values, electrostatic factors might also be involved in selectively excluding other negatively charged macromolecules transported through the interstitium (Fig. 1C). Data from the lung have indicated that fixed negative charges in the interstitial matrix significantly reduce the space available for anionic lactate dehydrogenase 1 (pI ~ 5.0) as compared to the cationic lactate dehydrogenase 5 (pI ~ 7.9) (Taylor & Parker, 2003), suggesting a charge effect on macromolecular probe distribution.

The magnitude of the excluded volume has important consequences in the dynamics of transcapillary exchange. Due to exclusion, the effective protein concentration in the interstitium is much higher than the value that would be estimated if it were assumed that all the fluid in the interstitium was available. As stated in the review by Aukland and Reed ( 1993), the physiological importance of the exclusion phenomenon is two-fold; its increase results in a more rapid approach to a new steady-state after a change in transcapillary fluid flow and less transfer of interstitial protein to plasma for a given capillary hyper-filtration. Interstitial exclusion thereby influences plasma volume regulation. Furthermore, the study of exclusion phenomena provides information regarding the organization of structural elements of the interstitium. Our recent in vitro and in vivo experiments involved testing the electrostatic exclusion hypothesis and, moreover, quantifying the magnitude of volume exclusion provided by these fixed negative charges in a given interstitium.

Together the interstitial fluid in skin and muscle accounts for almost 60% of the total body interstitial fluid volume (Aukland & Reed, 1993). Therefore these organs are of major importance for fluid balance studies, and have been the focus of our recent studies on the effects of charge on the distribution volume. Albumin is the most abundant plasma protein, and is an important determinant of plasma and interstitial colloid osmotic pressures.

Previous studies have stressed the importance of steric exclusion and shown that albumin is excluded from a large fraction of most interstitia (Aukland & Reed, 1993). Because of its pI of ~5 the albumin molecule has a net negative charge at physiological pH. Accordingly, this substance is a highly relevant candidate to use as probe in interstitial exclusion studies.

An important step for conducting our studies was the possibility of modifying the net charge of the anionic albumin to more positive values. Once this task was achieved we performed an in vitro study involving fully swollen rat dermis (Wiig et al. 2003). By quantifying the contribution of negative charges to the volume exclusion of albumin we found that a decrease in the net charge of albumin results in an increase in the interstitial distribution volume of this species. Thus, we demonstrated a substantial influence of negatively charged tissue elements on albumin distribution, amounting to about 40% of the albumin exclusion effect.

The in vivo experiments tested our hypothesis on albumin exclusion in skin and muscle of normally hydrated rats (Gyenge et al. 2003). A prerequisite for this type of study is to establish a steady-state tissue tracer concentration, which was done by continuous intravenous infusion of normal and cationized (net charge close to neutral) albumin for 5-7 days. Another requirement is to isolate fluid representative of interstitial fluid, and for this purpose we sampled fluid from wicks implanted in the tissues of interest.

As evident from Fig. 2, once again a net change in the charge of albumin significantly affected its exclusion volume in skin as well as muscle. From these data we were able to estimate that, on average, the contribution of fixed negative charges to albumin exclusion from skeletal muscle and skin interstitia is in the range of 25-40%, a contribution much higher than previously believed.

The findings discussed above may also have implications for therapy. With the emergence of new therapeutic tools like monoclonal antibodies, the distribution of macromolecular probes in tumours is of importance. Therefore, studies of exclusion phenomena are of considerable interest in tumours since the interstitium is a major barrier to drug delivery in this tissue (Jain, 1997). In preliminary studies in rat mammary tumours we have shown a significant effect of fixed negative charges on interstitial distribution of albumin, an effect that has to be considered when studying uptake of macromolecular therapeutic agents.

In conclusion, it thus seems that a greater exclusion of more negative proteins is a general phenomenon occurring in normal as well as pathological tissues.

References

Aukland K & Reed RK. (1993). Interstitial-lymphatic mechanisms in the control of extracellular fluid volume. Physiol Rev 73, 1-78.

Gyenge CC, Tenstad O & Wiig H. (2003). In vivo determination of steric and electrostatic exclusion of albumin in rat skin and skeletal muscle. J Physiol 552, 907-916.

Jain RK. (1997). The Eugene M. Landis Award Lecture 1996. Delivery of molecular and cellular medicine to solid tumors. Microcirculation 4, 1-23.

Ogston AG & Phelps CF. (1961). The partition of solutes between buffer solutions and solutions containing hyaluronic acid. Biochem J 78, 827-833.

Taylor AE & Parker JC. (2003). Intersitial exluded volumes: the effect of charge. J Physiol 553, 333.

Wiig H, Kolmannskog O, Tenstad O & Bert JL. (2003). Effect of charge on interstitial distribution of albumin in rat dermis in vitro. J Physiol 550, 505-514.