William M. Deen

Effects of norepinephrine and angiotensin II on the determinants of glomerular ultrafiltration and proximal tubule fluid reabsorption in the rat.

In 26 Wistar rats with surface glomeruli, the determinants of glomerular ultrafiltration and peritubular capillary uptake of proximal reabsorbate were studied before and during intravenous infusions of norepinephrine or angiotensin II. Regardless of whether renal perfusion pressure (AP) was permitted to increase, both hormones produced elevations in single nephron filtration fraction due to declines in glomerular plasma flow with little change in nephron glomerular filtration rate. The resulting large increases in the efferent arteriolar oncotic pressure, ±E, were accompanied by equivalent increases in the mean glomerular transcapillary hydraulic pressure difference, ±ΔP. Equality of ±E, and ±ΔP, denoting filtration pressure equilibrium, obtained before and during infusion of either hormone. For both hormones, when elevations in ±ΔP were allowed, marked and roughly proportional increases in the resistance to blood flow through single afferent and efferent arterioles occurred, whereas when increases in ±ΔP were prevented by partial aortic constriction increases in resistance were confined primarily to the efferent arteriole. Despite the marked increases in -±E, absolute rates of proximal tubule fluid reabsorption, on the average, were unchanged by these hormones due to the opposing effects of marked decreases in efferent arteriolar plasma flow rate and, to a lesser extent, increases in peritubular capillary hydraulic pressure.

Hindered diffusion in agarose gels: test of effective medium model

The diffusivities of uncharged macromolecules in gels (D) are typically lower than in free solution (D infinity), because of a combination of hydrodynamic and steric factors. To examine these factors, we measured D and D infinity for dilute solutions of several fluorescein-labeled macromolecules, using an image-based fluorescence recovery after photobleaching technique. Test macromolecules with Stokes-Einstein radii (rs) of 2.1-6.2 nm, including three globular proteins (bovine serum albumin, ovalbumin, lactalbumin) and four narrow fractions of Ficoll, were studied in agarose gels with agarose volume fractions (phi) of 0.038-0.073. The gels were characterized by measuring the hydraulic permeability of supported agarose membranes, allowing calculation of the Darcy permeability (kappa) for each gel sample. It was found that kappa, which is a measure of the intrinsic hydraulic conductance of the gel, decreased by an order of magnitude as phi was increased over the range indicated. The diffusivity ratio D/D infinity, which varied from 0.20 to 0.63, decreased with increases in rs or phi. Thus as expected, diffusional hindrances were the most severe for large macromolecules and/or relatively concentrated gels. According to a recently proposed theory for hindered diffusion through fibrous media, the diffusivity ratio is given by the product of a hydrodynamic factor (F) and a steric factor (S). The functional form is D/D infinity = F(rs/k1/2) S(f), where f = [(rs+rf)/rf]2 phi and rf is the fiber radius. Values of D/D infinity calculated from this effective medium theory, without use of adjustable parameters, were in much better agreement with the measured values than were predictions based on other approaches. The strengths and limitations of the effective medium theory for predicting diffusivities in gels are discussed.

DIFFUSION AND PARTITIONING OF PROTEINS IN CHARGED AGAROSE GELS

The effects of electrostatic interactions on the diffusion and equilibrium partitioning of fluorescein-labeled proteins in charged gels were examined using fluorescence recovery after photobleaching and gel chromatography, respectively. Measurements were made with BSA, ovalbumin, and lactalbumin in SP-Sepharose (6% sulfated agarose), in phosphate buffers at pH 7 and ionic strengths ranging from 0.01 to 1.0 M. Diffusivities in individual gel beads (D) and in the adjacent bulk solution (D infinity) were determined from the spatial Fourier transform of the digitized two-dimensional fluorescence recovery images. Equilibrium partition coefficients (phi) were measured by recirculating protein solutions through a gel chromatography column until equilibrium was reached, and using a mass balance. Diffusion in the gel beads was hindered noticeably, with D/D infinity = 0.4-0.5 in each case. There were no effects of ionic strength on BSA diffusivities, but with the smaller proteins (ovalbumin and lactalbumin) D infinity increased slightly and D decreased at the lowest ionic strength. In contrast to the modest changes in diffusivity, there were marked effects of ionic strength on the partition coefficients of these proteins. We conclude that for diffusion of globular proteins through gel membranes of like charge, electrostatic effects on the effective diffusivity (Deff = phi D) are likely to result primarily from variations in phi with only small contributions from the intramembrane diffusivity.

Dynamics of Glomerular Ultrafiltration in the Rat. IV. DETERMINATION OF THE ULTRAFILTRATION COEFFICIENT

Pressures and flow rates were measured in accessible surface glomeruli of mutant Wistar rats under conditions deliberately designed to prevent achievement of filtration pressure equilibrium, that is, the equalization of transcapillary hydrostatic and oncotic pressures by the efferent end of the glomerulus as typically observed in the normal hydropenic rat. Disequilibrium was obtained at elevated levels of glomerular plasma flow (GPF) brought about by acute expansion of plasma volume with a volume of rat plasma equal to 5% of body weight. Glomerular hydrostatic and oncotic pressures measured at high GPF were used to calculate the ultrafiltration coefficient, K(f), the product of effective hydraulic permeability and surface area. GPF was then either lowered (by aortic constriction) or raised (by carotid occlusion) in order to examine the dependence of K(f) on GPF. The value of K(f) per glomerulus, 0.08 nl/(s.mm Hg), was found not to vary over an approximately twofold range of GPF. This finding, taken together with data from previous studies from this laboratory, leads us to conclude that plasma-flow dependence of glomerular filtration rate (GFR) results primarily from flow-induced changes in mean ultrafiltration pressure, rather than large changes in K(f).

Determination of serum protein concentration in nanoliter blood samples using fluorescamine or o-phthalaldehyde

Fluorometric procedures were developed to permit measurement of total protein concentration in nanoliter serum samples, using either fluorescamine or o-phthalaldehyde. The sensitivities of assays using these two reagents were similar, but the o-phthalaldehyde method was found to be somewhat simpler and more reproducible. Accurate measurements could be obtained on serum samples of 4 to 5 nl with either reagent, by using a serum standard. Fluorescence differed considerably among individual serum proteins, albumin generally showing greater fluorescence than globulins. Small molecular weight species in serum did not contribute appreciably to total serum fluorescence with either reagent.

What determines glomerular capillary permeability

There have been exciting recent advances in our understanding of the structural and molecular biology of the glomerular slit diaphragm, as described in a report in this issue of the JCI. These findings, combined with data on the permeability of the basement membrane and evidence that the endothelium may be a more important barrier than often supposed, are allowing a clearer understanding to emerge of how the 3 parts of the glomerular capillary wall jointly determine its functional properties.

Diffusivity and solubility of nitric oxide in water and saline.

Aqueous diffusivities and solubilities of NO were determined by using a chemiluminescence detector to measure time-dependent fluxes across stagnant liquid films confined between polydimethylsiloxane membranes. The NO diffusivities in pure water and PBS at 25∘C were found to be (2.21 ± 0.02) × 10−5 cm2 s−1 and (2.21 ± 0.04) × 10−5 cm2 s−1, respectively. Although lower than most previous values for NO at room temperature, these diffusivities are very similar to those for O2, as one would expect. Extrapolation to 37°C yielded a value of 3.0 × 10−5 cm2 s−1. The solubility of NO in water at 25°C was (1.94 ± 0.03) × 10−6 mol cm−3 atm−1, in agreement with the literature. This agreement, along with the excellent fits obtained to the transient flux data (<4% rms error in each experiment), supports the validity of the simultaneously measured diffusivity. The solubility of NO in PBS at 25°C was (1.75 ± 0.02) × 10−6 mol cm−3 atm−1. The modest (10%) reduction in solubility relative to that in pure water is consistent with the usual effects of salts on gas solubilities.

Electrostatic effects on the partitioning of spherical colloids between dilute bulk solution and cylindrical pores

Abstract Theoretical results are presented for the electrostatic double-layer interaction between a colloid particle and a long cylindrical pore, where the colloid is allowed to assume any radial position within the pore. The colloid particle is represented as a solid sphere with a given surface charge density or surface potential, or as a porous sphere with a given volumetric charge density, and the linear form of the Poisson-Boltzmann equation is employed. Analytical expressions for the potential energy of interaction are obtained and used to calculate equilibrium partitioning coefficients.

Electrostatic double-layer interactions for spherical colloids in cylindrical pores

Abstract Theoretical results are presented for the electrostatic double-layer interaction between a colloid particle and a long cylindrical pore, where the colloid is represented as a solid sphere with a given surface charge density or surface potential or as a porous sphere with a given volumetric charge density. The analysis is restricted to axisymmetric positions of the sphere and the linear form of the Poisson—Boltzmann equation is employed. Analytical expressions for the potential energy of interaction are obtained, and application of these to the calculation of equilibrium partitioning coefficients is discussed.

Hindered transport in fibrous membranes and gels: Effect of solute size and fiber configuration

Abstract Hindered transport coefficients for a spherical macromolecule in a spatially periodic fibrous medium were calculated using two different methods. The first method is an effective medium approach based on Brinkmans equation and can be readily applied to disordered fibrous media. The second and more rigorous set of calculations makes use of generalized Taylor dispersion theory. Results from these two approaches are compared for two different spatially periodic lattices of bead-and-string fibers, which were chosen to illustrate the effects of inhomogeneity in the distribution of fibers. In addition, ratios of solute radius to fiber radius ranging from 0.5 to 5.0 were considered. Qualitative agreement between the two methods was obtained for each case studied, and quantitative agreement was obtained for volume fractions at which the hindering effects of the fibers were not too severe.