Dextran

ABSTRACT Novel agarose-dextran hydrogels were synthesized and their suitability as experimental models of glomerular basement membrane was examined by measuring their Darcy (hydraulic) permeabilities (kappa). Immobilization of large dextran molecules in agarose was achieved by electron beam irradiation. Composite gels were made with agarose volume fractions (phi^sub a^) of 0.04 or 0.08 and dextran volume fractions (phi^sub a^) ranging from 0 to 0.02 (fiber volume/gel volume), using either of two dextran molecular weights (500 or 2000). At either agarose concentration and for either size of dextran, kappa decreased markedly as the amount of dextran was increased. Statistically significant deviations from the value of K for pure agarose were obtained for remarkably small volume fractions of dextran: phi^sub d^ - 0.0003 for phi^sub a^ = 0.04 and phi^sub a^ >= 0.001 for phi^sub a^ = 0.08. The Darcy permeabilities were much more sensitive to phi^sub d^ than to phi^sub a^, and were as much as 26 times smaller than those of pure agarose. Although phi^sub d^ was an important variable, dextran molecular weight was not. The effects of dextran addition on K were described fairly well using simple structural idealizations. At high agarose concentrations, the dextran chains behaved as fine fibers interspersed among coarse agarose fibrils, whereas, at low concentrations, the dextran molecules began to resemble spherical obstacles embedded in agarose gels. The ability to achieve physiologically relevant Darcy permeabilities with these materials (as low as 1.6 nm^sup 2^) makes them an attractive experimental model for glomerular basement membrane and possibly other extracellular matrices.

INTRODUCTION

Pressure-driven flow through extracellular matrices is important in the normal function or pathophysiology of a variety of tissues, including cartilage (Treppo et al., 2000), arterial intima (Baldwin and Wilson, 1993), juxtacanalicular tissue of the eye (Johnson et al., 1992), and tumor interstitium (Netti et al., 2000). Of particular interest to our laboratory is water flow through the glomerular basement membrane (GBM). Flow through that structure is crucial for renal function, in that the ultrafiltration of blood across the walls of glomerular capillaries, which are composed of a layer of fenestrated endothelial cells, the GBM, and a layer of epithelial foot processes, is the first step in urine formation. Two hallmarks of chronic kidney disease are progressive reductions in the glomerular filtration rate of water, and increases in the amounts of protein that are filtered and appear in urine (Blouch et al., 1997; Deen et al., 2001). Inasmuch as the GBM significantly influences both the water-filtration capacity of glomeruli and their ability to selectively retain proteins or other macromolecules in the circulation, there is considerable motivation to measure and explain its transport properties.

The isolation of GBM from rats or other experimental animals, using dissection, sieving to collect whole glomeruli, and detergent lysis to remove cells, has made it possible to measure its hydraulic and macromolecular permeabilities in vitro (Robinson and Walton, 1987; Walton et al., 1992; Daniels et al., 1992, 1993; Edwards et al., 1997; Bolton et al., 1998). Such studies provide the foundation for our current understanding of the contribution of the GBM to the overall permeability properties of the glomerular capillary wall (Deen et al., 2001). However, they yield little insight into the relationship between the functional properties and macromolecular structure of GBM, because it has not been possible to systematically vary (or even fully quantify) the composition of isolated GBM. Moreover, technical limitations have precluded making independent measurements of diffusive and convective hindrance factors for proteins or other macromolecules using the same GBM preparation. The ability to create membranes of desired dimensions and composition has made it attractive to investigate polymeric hydrogels as experimental models. For example, poly(glyceryl methacrylate) gels have been proposed as synthetic analogs of GBM (Leung and Robinson, 1992).

In considering what a synthetic hydrogel should mimic, we note that GBM consists mainly of collagen IV, laminin, and glycosaminoglycans (GAGs) such as heparan sulfate (Yurchenco and Schittny, 1990), and that approximately 90% of its volume is water (Robinson and Walton, 1987; Comper et al. 1993). Collagen IV and laminin have been shown to self-assemble into three-dimensional networks, and are thought to form primary and secondary frameworks in the GBM. Micrographs of collagen IV networks formed in vitro show fibrils ~1.5-6 nm thick (Yurchenco and Ruben, 1988; Yurchenco and Schittny, 1990), whereas those of laminin networks show fibrils ~4 nm thick (Yurchenco et al., 1992). Both fibril sizes are comparable to typical thicknesses seen in micrographs of GBM (6-8 nm; Bolton and Deen, 2002), suggesting that it is these protein networks (and not the GAGs) that are visualized. Heparan sulfate is an extended, flexible chain of ~1 nm in diameter (Dea et al., 1973). Unlike collagen IV and laminin, heparan sulfate does not appear to form its own network; instead, it attaches to the collagen IV network through disulfide bonds (Parthasarathy and Spiro, 1981). Thus, GBM may be viewed as consisting of at least two types of fibers (coarse and fine), which together comprise about 10% of its volume.

The representation of GBM as a composite gel containing two types of fibers is supported by analyses of its permeability properties measured in vitro (Edwards et al., 1997; Bolton and Deen, 2002). It was found that theories based on a uniform population of fibers were incapable of reconciling the measured hydraulic (or Darcy) permeability, the reported GBM solids content, and the fiber radii observed in micrographs. Only when a substantial number of smalldiameter fibers were introduced in the calculations was it possible to account for the relatively low Darcy permeability of GBM (1-2 nm^sup 2^). Likewise, it was found that a one-fiber representation did not adequately explain the sieving coefficients measured for neutral, spherical test solutes (Ficoll) of various sizes across isolated GBM (Bolton and Deen, 2002). Thus, a mixture of fiber types is suggested both by the biopolymers present and by the known permeability properties.

Agarose gels are an attractive starting point for creating an experimental model of the GBM, because they contain fibrils with diameters of ~4 nm, roughly comparable to those of the relevant protein networks. They are readily made, and have a cross-linked network of fibrils that is rigid enough to exhibit negligible osmotic shrinkage when they are exposed to concentrated solutions of macromolecules (White and Deen, 2001). Moreover, the water permeability and the hindered diffusion and convection of macromolecules have been relatively well studied in agarose gels (Johnson et al., 1995, 1996; Johnson and Deen, 1996; Kong et al., 1997; Pluen et al., 1999; Johnston and Deen, 1999, 2002). Neutral dextran is a reasonable choice for the second polymeric component, because it too is readily available, and its backbone diameter approximates that of heparan sulfate. Although dextran is uncharged and heparan sulfate is a polyanion, the absence of measurable charge selectivity in the filtration of macromolecules across isolated GBM at physiological ionic strength (Bolton et al., 1998) suggests that this difference is not crucial. Accordingly, the objective of the present study was to synthesize agarose-dextran composite hydrogels and to examine their suitability as experimental models of GBM. Immobilization of dextran in agarose gels was achieved by electron beam irradiation. Darcy permeabilities were measured as a function of the relative amounts of agarose and dextran, and the results compared with predictions from various theories for water flow through fibrous media.

METHODS

Materials

Agarose (type VI, high-gelling) and unlabeled and FITC-labeled dextrans with nominal (weight-averaged) molecular weights (M^sub w^) of 500 or 2000 were purchased from Sigma Chemicals (St. Louis, MO). Before use, solutions of FITC dextran were ultrafiltered across Biomax membranes (50 kDa nominal molecular mass cutoff, Millipore, Bedford, MA) to verify that the amount of free fluorescein was negligible. Polyester meshes (70 (mu)m thick, 53% open area), used as supports for the gel membranes, were purchased from Spectrum Laboratories (Houston, TX). The buffer solutions used were 0.01 M sodium phosphate at pH 7, with 0.02% sodium azide added as a bactericide.

Gel synthesis

Agarose-dextran composite gets were synthesized by preparing agarose gels, equilibrating them with dextran solutions, and using electron-beam irradiation to covalently link the dextran to the agarose fibrils. The variables examined were agarose volume fraction, dextran concentration, and dextran molecular weight. Agarose gels with volume fractions (phi^sub a^) of 0.04, 0.06, or 0.08 were prepared by first dissolving agarose in phosphate buffer at pH 7 in an oven at 95 deg C. (The volume fraction of agarose is essentially the same as its mass concentration in g/ml.; Johnson et al., 1995.) The solutions were allowed to gel at room temperature. The agarose gels were equilibrated with solutions of 2-15% (w/v) of either size of dextran. Relatively large volumes of buffer (~300 X gel volume) were used to incubate the gets, so that the dextran concentration in the buffer would be constant. To ensure equilibrium, the incubation periods equaled several characteristic diffusion times, tau^sub d^. Values for tau^sub d^, estimated using the data of Key and Sellen (1982) for diffusion of 500 and 2000 kDa dextran in agarose, ranged from ~30 min to 5 hr for a gel thickness of 100 (mu)m.

Electron-beam irradiation links dextran and agarose by using OH radicals formed by the introduction of electrons into the aqueous phase to abstract hydrogen radicals from both dextran and agarose. When a resulting carbon radical from one chain encounters one from another, a cross-link forms. All results presented here are for samples irradiated for ~1 s at a dosage of 2 Mrad, at the High Voltage Research Laboratory at Massachusetts Institute of Technology. In preliminary experiments, several samples (with and without dextran) were irradiated at doses ranging from I to 4 Mrad. Within that range, there was no significant effect of dosage on the measured water permeabilities.

Dextran concentrations in gels

Dr. Edward W. Merrill suggested the use of electron beam irradiation to covalently link dextran to agarose, and Mr. Kenneth A. Wright provided invaluable assistance in exposing the gels to the beam.

This work was supported by grant DK20368 from the National Institutes of Health. J.A.W. is the recipient of a National Science Foundation Graduate Fellowship.

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Jeffrey A. White* and William M. Deen*,^

*Department of Chemical Engineering and ^Division of Bioengineering and Environmental Health, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 USA

Submitted June 18, 2001, and accepted for publication January 2, 2002.

Address reprint requests to William M. Deen, Dept. of Chemical Engineering, 66-572, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139. Tel.: 617-253-4535; Fax: 617-258-8224; E-mail: wmdeen@mit.edu

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