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W M Deen

Publications and source records attributed to W M Deen.

At least 37 records · Page 2Linked to original sources
PubMed1996

Kinetics of S-nitrosation of thiols in nitric oxide solutions.

The S-nitroso adducts of nitric oxide (NO) may serve as carriers of NO and play a role in cell signaling and/or cytotoxicity. A quantitative understanding of the kinetics of S-nitrosothiol formation in solutions containing NO and O2 is important for understanding these roles of S-nitroso compounds in vivo. Rates of S-nitrosation in aqueous solutions were investigated for three thiols: glutathione, N-acetylcysteine, and N-acetylpenicillamine. Nitrous anhydride (N2O3), an intermediate in the formation of nitrite from NO and O2, is the most likely NO donor for N-nitrosation of amines as well as for S-nitrosation of thiols, at physiological pH. This motivated the use of a competitive kinetics approach, in which the rates of thiol nitrosation were compared with that of a secondary amine, morpholine. The kinetic studies were carried out with known amounts of NO and O2 in solutions containing one thiol (400 microM) and morpholine (200-5700 microM) in 0.01 M phosphate buffer at pH 7.4 and 23 degrees C. It was found that disulfide formation, transnitrosation reactions, and decomposition of the S-nitrosothiols was expressed as k7[N2O3][RSH], where RSH represents the thiol. The rate constant for S-nitrosation relative to that for N2O3 hydrolysis (k4) was found to be k7/k4 = (4.15 +/- 0.28) x 10(4), (2.11 +/- 0.11) x 10(4), and (0.48 +/- 0.04) x 10(4) M-1 for glutathione, N-acetylcysteine, and N-acetylpenicillamine, respectively. The overall (observed) rates of nitrosothiol formation reflect the fact that [N2O3] varies [NO]2[O2] and that [N2O3] also depends on [RSH] and the concentration of phosphate. Using a detailed kinetic model to account for these effects, the present results could be reconciled with apparently dissimilar findings reported previously by others.

Acetylcysteine↗
PubMed1995

Kinetic analysis of the fate of nitric oxide synthesized by macrophages in vitro.

To investigate the fate of nitric oxide (NO) synthesized by activated macrophages, the concentrations of NO and its principal reaction products, nitrite (NO2-) and nitrate (NO3-), were measured as a function of time in suspension cultures of RAW264.7 macrophages attached to microcarrier beads. Synthesis of NO became evident 2-5 h after stimulation of the cells, and steady concentrations of NO were achieved after about 9 h. The appearance of NO in the extracellular fluid coincided with the appearance of NO2- and NO3-, which were formed thereafter at approximately equal and constant rates. Using a kinetic model based on rate constants measured previously in cell-free systems, only half of the NO2- formed could be accounted for by the reaction of NO with O2. It is known that NO reacts with superoxide (O2.) to give peroxynitrite and that NO also reacts with peroxynitrite to yield NO2-, so that the latter reaction may explain the "excess" NO2- formation. Adding superoxide dismutase to the medium markedly reduced the ratio of NO3- to NO2-, consistent with the hypothesis that NO3- in the medium results primarily from the extracellular reaction of NO with O2-.. The addition of morpholine, a model amine, resulted in formation of N-nitrosomorpholine, concurrent with the other products. Measured rates of nitrosomorpholine formation were 6-fold lower than predictions based on kinetics in simple solutions, suggesting that in the cell culture system there were additional reactions that lowered the concentration of nitrous anhydride, the principal nitrosating agent formed from NO and O2.

Cell Line↗
PubMed1995

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.

Animals↗
PubMed1995

Hindered transport of macromolecules through a single row of cylinders: application to glomerular filtration.

The slit diaphragms of renal glomerular capillaries form an ultrafiltration barrier which may be approximated as a row of cylindrical fibers of macromolecular dimensions. To describe the hindered transport of plasma proteins and other macromolecules through this barrier, we developed an approximate hydrodynamic model for spherical, Brownian particles passing through a row of infinitely long cylinders. The selectivity of the slit diaphragm was assessed by computing concentration profiles for a wide range of molecular sizes for Pe < or = 1, where Pe is a Peclet number based on the cylinder radius. The sieving coefficient for the slit diaphragm was computed as theta SD = CB/CO, where CO was the average concentration at a specified distance upstream from the cylinders (corresponding to the location of the basement membrane), and CB was the concentration far downstream (corresponding to Bowman's space). The results of previous experimental sieving studies using rats could be accounted for approximately by postulating a wide distribution of spacings between the fibers of the slit diaphragm. Comparing the results for theta SD with calculations for a model of the glomerular basement membrane suggests that the slit diaphragm is by far the more size-restrictive part of the overall barrier.

Animals↗
PubMed1995

Error propagation in the estimation of glomerular pressure from macromolecule sieving data.

The theoretical effects of the glomerular transmural hydraulic pressure difference (delta P) on the sieving coefficients (theta i) of macromolecules of varying size have led to a number of attempts to use sieving curves to estimate delta P noninvasively, with inconsistent results. The objective of this study was to determine the extent to which experimental errors and imperfections in the theoretical models limit the ability to obtain reliable estimates of delta P using this method. Our approach was to generate many sets of synthetic "experimental data" using computer simulations of glomerular sieving and to compute values of delta P by fitting models to those data in the presence of various types and magnitudes of errors. Unbiased experimental errors were simulated by adding random amounts to individual values of theta i, and systematic errors were investigated by using a model based on one type of pore-size distribution to fit "data" generated using a model of a different type. We found that with random errors in theta i only, the estimate of delta P was accurate to within +/- 4 mmHg nearly all of the time, provided that the standard deviation, sigma i, was < or = 5% of theta i. When there were also systematic errors arising from the use of an "incorrect" form of pore-size distribution, a useful predictor of success was the probability P that the residuals, the differences between the measured and predicted sieving coefficients, were randomly distributed. A value of P > 0.2, as calculated from the algebraic signs of the residuals, indicated a high likelihood that the pressure estimate was accurate, provided that the random errors were sufficiently small. When P > 0.2, the fitted value of delta P was within +/- 4 mmHg of the true value in about 90%, 80%, and 70% of the cases examined when sigma i was < or = 2%, 5%, or 10% of theta i, respectively. An analysis of published data from a number of experimental studies indicated, however, that the favorable conditions of small sigma i and large P are extremely difficult to achieve, making it unlikely that an accurate group-mean value of delta P will be estimated from any given set of sieving data. Significant experimental and theoretical advances will be needed to make this a reliable method for estimating glomerular pressure.

Animals↗
PubMed1994

Metabolism and excretion of methylamines in rats.

Experiments were performed with rats to examine the sources and disposition of dimethylamine (DMA), trimethylamine (TMA), and trimethylamine N-oxide (TMAO), all potential substrates for in vivo nitrosation to form N-nitrosodimethylamine (NDMA), a potent carcinogen. When bolus doses of [14C]DMA or [14C]TMA were given ip, recovery of radioactivity in the urine was essentially complete, and respiratory excretion, fecal excretion, and accumulation in tissues of these amines or their metabolites were negligible. Urine analysis following doses of stable isotopes showed that DMA was not converted to TMA or TMAO. Varying amounts of TMA were oxidized to TMAO, the fraction oxidized decreasing at higher doses of TMA. Ingestion and excretion of naturally occurring methylamines were monitored over a 5-day period in separate groups of normal and germ-free rats. The results of these metabolic balance studies indicate that there is net synthesis of DMA by gut bacteria and net consumption of TMAO by endogenous processes. The net intake or excretion of TMA and TMAO observed in normal and germ-free rats is consistent with bacterial synthesis of TMA followed by its almost complete oxidation to TMAO. Blood concentrations of DMA and TMA were measured in rats for 8 hr following < 5, 100, or 1000 mumol bolus i.v. or ip doses of radioisotopes or stable isotopes. At any given dose of DMA or TMA, the decay in blood concentration was approximately monoexponential. At the lowest (most physiologic) dose the apparent volume of distribution (VD) for DMA was larger than that for TMA. Both values of VD greatly exceeded the size of the animals, suggesting that DMA and TMA are highly concentrated at one or more locations in the body. This was confirmed by measurements in tissue homogenates sampled 1 hr after a dose. The overall handling of methylamines by rats is generally consistent with observations in humans. The presence of high local concentrations of DMA and TMA at various extragastric sites merits further investigation in connection with the potential for endogenous nitrosation of these methylamines to form NDMA.

Animals↗
PubMed1994

Random-coil model for glomerular sieving of dextran.

Dextran has been the most commonly employed test molecule for probing the selectivity of glomerular filtration to macromolecules of varying size. The usual theories for hindered transport of solid spheres through pores have limited utility in interpreting clearance data for dextran or other linear polymers because such polymers in solution more closely resemble random, solvent-filled coils than solid spheres. To provide a model for glomerular filtration of random-coil macromolecules, the equilibrium partitioning of random coils between cylindrical pores and bulk solution was simulated using Monte Carlo calculations, and those results were combined with a hydrodynamic theory for restricted motion of solvent-filled polymer coils in pores. The rates of transport predicted for either neutral random coils or for solid spores of the same Stokes-Einstein radius were significantly lower than observed transport rates of dextran through the glomerular capillary wall or across synthetic porous membranes. This facilitation of dextran transport was modeled by postulating weak, attractive interactions between dextran monomers and the pore wall. The random-coil model with attractive interactions, modeled using a short-range, square-well potential, was found to adequately represent dextran sieving data in normal rats. Various limitations of this approach are discussed.

Animals↗
PubMed1994

Stokes flow through a row of cylinders between parallel walls: model for the glomerular slit diaphragm.

As a model for flow through the slit diaphragms which connect the epithelial foot processes of renal glomerular capillaries, finite element solutions of Stokes equations were obtained for flow perpendicular to a row of cylinders confined between parallel walls. A dimensionless "additional resistance" (f), defined as the increment in resistance above the Poiseuille flow value, was computed for L/W < or = 4 and 0.1 < or = R/L < or = 0.9, where L is half the distance between cylinder centers, W is half the distance between walls and R is the cylinder radius. Two factors contributed to f: the drag on the cylinders, and the incremental shear stresses on the walls of the channel. Of these two factors, the drag on the cylinders tended to be dominant. A more complex representation of the slit diaphragm, suggested in the literature, was also considered. The predicted hydraulic permeability of the slit diaphragm was compared with experimental values of the overall hydraulic permeability of the glomerular capillary wall.

Capillary Permeability↗
PubMed1994

Structural determinants of glomerular hydraulic permeability.

To elucidate which structures determine the resistance to water movement, we used a computational fluid dynamics approach to determine velocity and pressure fields within the glomerular capillary wall. The model included representations of the endothelial fenestrae, basement membrane, and epithelial filtration slits with slit diaphragms. The input data included dimensions of the various structures from previous electron microscopy studies, as well as the hydraulic permeability recently measured for isolated films of glomerular basement membrane in vitro. The hydraulic resistance of the endothelium was predicted to be small, whereas the basement membrane and filtration slits were each found to contribute roughly one-half of the total hydraulic resistance of the capillary wall. It was calculated that, for a given filtrate flux, the pressure drop within basement membrane in vivo is roughly twice that of "bare" or isolated basement membrane, because of the small fraction of basement membrane area exposed. The dominant resistance in the filtration slit was found to be the slit diaphragm. Predicted values for the overall hydraulic permeability of the capillary wall were within the experimental range derived from micropuncture measurements in normal rats. The model should be a useful tool for analyzing the effects of various structural changes on glomerular hydraulic permeability. This is illustrated by applying the model to recent physiological and morphometric data in nephrotic rats.

Animals↗
PubMed1994

Proteinuria and impaired glomerular permselectivity in uninephrectomized fawn-hooded rats.

Previous studies of glomerular permselectivity have indicated that both size selectivity and charge selectivity changes play a role in the pathogenesis of proteinuria. In this study, we measured Ficoll sieving coefficients, hemodynamic parameters, and urinary protein excretion rates in the FHH strain of fawn-hooded rats. These animals spontaneously develop systemic and glomerular hypertension, proteinuria, and focal and segmental glomerulosclerosis at a relatively young age. Three groups of FHH rats were studied: two-kidney controls (2K), untreated uninephrectomized rats (CON-NX), and uninephrectomized rats treated with the angiotensin I converting enzyme inhibitor enalapril (ENA-NX). CON-NX rats had higher glomerular transcapillary pressures (delta P) and higher urinary excretion rates of both total protein (UpV) and albumin (UaV) than did 2K rats, whereas treatment with enalapril prevented both glomerular hypertension and the increased proteinuria. Ficoll sieving coefficients were significantly higher in both groups of NX rats compared with 2K rats only for Stokes-Einstein radii (rs) > or = 46 A. Fits of sieving data to pore models showed a small increase in the number of large, nonselective pores in NX, which was not prevented by enalapril treatment. Total clearances of Ficoll with rs = 36 A (the size of albumin) in CON-NX and ENA-NX groups were unchanged compared with 2K animals. In contrast, UaV in CON-NX rats was more than six times that of 2K and ENA-NX rats. Across groups, UpV, UaV, and the ratio (UaV)/(UpV) all correlated strongly with delta P.(ABSTRACT TRUNCATED AT 250 WORDS)

Albuminuria↗
PubMed1994

Structural basis for reduced glomerular filtration capacity in nephrotic humans.

Previous studies have established that in a variety of human glomerulopathies the reduced glomerular filtration rate (GFR) is due to a marked lowering of the ultrafiltration coefficient (Kf). To identify the factors which lower Kf, we measured the filtering surface area per glomerulus, filtration slit frequency, basement membrane thickness, and GFR and its determinants in patients with minimal change and membraneous nephropathies and in age-matched healthy controls. Overall values of Kf for the two kidneys were calculated from GFR, renal plasma flow rate, systemic colloid osmotic pressure, and three assumed values for the transcapillary pressure difference. "Experimental" values of the glomerular hydraulic permeability (kexp) were then calculated from Kf, glomerular filtering surface area, and estimates of the total number of nephrons of the two kidneys. Independent estimates of the glomerular hydraulic permeability (kmodel) were obtained using a recent mathematical model that is based on analyses of viscous flow through the various structural components of the glomerular capillary wall. Individual values of basement membrane thickness and filtration slit frequency were used as inputs in this model. The results indicate that the reductions of Kf in both nephropathies can be attributed entirely to reduced glomerular hydraulic permeability. The mean values of kexp and kmodel were very similar in both disorders and much smaller in the nephrotic groups than in healthy controls. There was good agreement between kexp and kmodel for any given group of subjects. It was shown that, in both groups of nephrotics, filtration slit frequency was a more important determinant of the water flow resistance than was basement membrane thickness. The decrease in filtration slit frequency observed in both disorders caused the average path length for the filtrate to increase, thereby explaining the decreased hydraulic permeability.

Adolescent↗
PubMed1993

Membrane mass spectrometer inlet for quantitation of nitric oxide.

Nitric oxide (NO) is an important physiological and biochemical messenger that may be involved in endogenous carcinogenesis and cell toxicity via formation of N-nitroso compounds or direct DNA damage by nitrosating agents arising from the reaction of NO with O2. To study the reaction of NO with O2 in model systems and the formation and disappearance of NO in more physiological systems such as cell cultures, we adapted and optimized a membrane mass spectrometer inlet specifically for such analyses. The inlet consisted of Silastic tubing inserted into a Swagelok 'tee', which was attached to the mass spectrometer via the tuning probe. Kinetics of NO disappearance can be followed under electron impact conditions until NO2 interferes via the formation of NO+ during fragmentation of NO2+. The aqueous NO concentration for minimum detection was determined to be 1.4 microM. The inlet response time to step changes in aqueous NO concentrations was 7.0 s, fast enough to permit real-time measurements of aqueous NO changes upon addition of O2. Finally, the depletion of aqueous NO was observed in the presence of O2. The relative steady state responses of inlets designed for gas or aqueous samples, and their relative response times, are explained by an analysis based on mass transfer theory.

Kinetics↗
PubMed1993

Effects of angiotensin II receptor blockade on remnant glomerular permselectivity.

This study examined the mechanisms by which angiotensin II (Ang II) receptor blockade improves glomerular barrier function in rats with reduced nephron number. Proteinuria was measured at four weeks after 5/6 renal ablation, and rats were then divided into a group which received the Ang II receptor blocker MK954 and a group which received no treatment. Studies performed one week later showed that Ang II receptor blockade reduced proteinuria without altering GFR in renal ablated rats. Micropuncture studies showed that Ang II blockade reduced both mean arterial pressure (142 +/- 7 mm Hg, ablation without treatment; 105 +/- 2 mm Hg, ablation with treatment) and glomerular transcapillary pressure (54 +/- 3 mm Hg, ablation without treatment; 43 +/- 1 mm Hg, ablation with treatment). Dextran sieving studies showed that untreated rats developed a size-selective defect characterized by increased transglomerular passage of neutral dextrans with radii 54 to 76 A and a charge-selective defect characterized by an increased transglomerular passage of anionic dextran sulfate with a radius of approximately 18 A. Ang II blockade reduced fractional clearance values for large neutral dextrans near to values observed in normal rats but had no effect on the fractional clearance of dextran sulfate (0.68 +/- 0.11, ablation without treatment; 0.66 +/- 0.08, ablation with treatment; 0.46 +/- 0.05, normal rats). These findings indicate that reducing Ang II activity improves size-selectivity without affecting charge-selectivity in injured remnant glomeruli.

Angiotensin II↗
PubMed1993

Glomerular permeability barrier in the rat. Functional assessment by in vitro methods.

The formation of glomerular ultrafiltrate is dependent on the prevailing hemodynamic forces within the glomerular microcirculation and the intrinsic properties of the filtration barrier. However, direct assessment of the permeability barrier is difficult with most available techniques. We used confocal microscopy to image 1-micron thick optical cross-sections of isolated intact glomeruli and glomeruli denuded of cells and quantitated dextran (70,000 mol wt) diffusion from the capillary lumen. Dextran permeance was 11 times greater for the acellular filtration barrier than the intact peripheral capillary. Consideration of the basement membrane and cells as series resistors demonstrated that cells of the filtration barrier contribute 90% of the total resistance to macromolecular permeance. Using a different approach, dextran sieving coefficients for acellular glomeruli consolidated as a multilayer sheet in a filtration cell were similar to those for intact glomeruli in vivo at radii 30-36 A and approximately 50 times greater at a dextran radius of 60 A. The presence of cells significantly reduced hydraulic permeability determined on consolidated intact or acellular glomeruli in an ultrafiltration cell with 50 mmHg applied pressure. The glomerular basement membrane does restrict macromolecular permeability but cells are important determinants of the overall macromolecular and hydraulic permeability of the glomerulus.

Animals↗
PubMed1993

Charge selectivity of the glomerular filtration barrier in healthy and nephrotic humans.

We used dextran sulfate (DS) to evaluate barrier charge selectivity in 11 nonproteinuric subjects and in 11 patients with the nephrotic syndrome due to either membranous nephropathy or minimal change nephropathy. The 3H-DS preparation spanned a molecular radius interval of 10-24 A and exhibited size-dependent protein binding in vitro. Urine and ultrafiltrates of plasma were separated by size into narrow fractions using gel permeation chromatography. The sieving coefficient (theta) for ultrafilterable DS of 15A radius averaged 0.68 +/- 0.03 in nonproteinuric vs. 0.95 +/- 0.05 in nephrotic subjects (P < 0.001). Uncharged dextrans of broad size distribution were used to evaluate barrier size-selectivity in separate groups of nonproteinuric subjects (n = 19) and nephrotic patients with either minimal change (n = 20) or membranous nephropathy (n = 27). The value of theta for an uncharged dextran of similarly small radius (approximately 18 A) was significantly larger than that observed for DS in nonproteinuric subjects, but was similar in nephrotic individuals. Further, impaired barrier size-selectivity, as assessed by the sieving profile for uncharged dextrans (18-60 A radius), failed to account fully for the observed level of albuminuria in almost half of the patients with either minimal change (9/20) or membranous nephropathy (12/27). Together these findings suggest that the human glomerular capillary wall normally provides an electrostatic barrier to filtration of negatively charged macromolecules such as albumin, and that impairment of this electrostatic barrier contributes to the magnitude of albuminuria in the nephrotic syndrome.

Adult↗
PubMed1992

Glomerular basement membrane: in vitro studies of water and protein permeability.

The glomerular basement membrane (GBM) is an integral structural component of the glomerular filter, but its contribution to the hydraulic and macromolecular permeability properties of the glomerulus has been the subject of much controversy. We have modified previously reported methods to develop a technique with which to study filtration properties of microgram quantities of isolated GBM in vitro at physiological pressures. Rat glomeruli were sieved, and cells were removed with N-laurylsarcosine and DNase. GBM (150 micrograms; greater than 95% pure) were added to a mini-ultrafiltration cell and consolidated under pressure to form a continuous filter at the base of the cell. Water flux was identical to inulin clearance at applied pressures less than 150 mmHg and increased with progressive increments in the transmembrane pressure. Hydraulic conductivity of GBM was inversely related to the prevailing transmembrane pressure gradient. The hydraulic conductivity depended on albumin concentration in a manner that was not monotonic, with the conductivity being lower at 4 g/dl albumin than at 0 or 8 g/dl. When plasma was utilized as the retentate, the fractional clearance of albumin was over twice that of immunoglobulin G, and the fractional clearance of each protein was much higher than that in the intact glomerulus. On the basis of these results, both the hydraulic and macromolecular permeability of an individual layer of GBM are much greater than that reported for the intact glomerulus. The large quantitative differences between GBM permeability and that of intact glomeruli suggest a major contribution of cellular elements to glomerular permeability properties.

Animals↗
PubMed1992

Filtration dynamics and natriuretic response to volume expansion in humans.

We used differential solute clearances and a theoretical analysis of glomerular ultrafiltration and dextran sieving to characterize the hemodynamic response of nine healthy humans to infusion of isoncotic, 5% albumin in saline or saline vehicle alone. During albumin infusion (10.2 +/- 0.2 ml.kg-1.30 min-1) plasma volume increased by 18%, but oncotic pressure rose by only 0.8 mmHg. Despite the hypervolemia, renal blood flow (RBF) declined by 140 ml/min and glomerular filtration rate (GFR) declined by 16 ml/min during the infusion. RBF increased progressively postinfusion, exceeding baseline by 135 ml/min after 4 h; GFR was restored to baseline. Although oncotic pressure declined by 2 mmHg, a similar transient decline in GFR (-13 ml/min) was associated also with infusion of saline vehicle alone (9.4 +/- 0.3 ml.kg-1.30 min-1), which increased plasma volume by 9%. Sieving coefficients of dextrans (radius 32-42 A) were lowered during and after either infusion, a phenomenon that we compute to reflect a reduction in glomerular pore size. Assuming that the transcapillary hydraulic pressure difference was not lowered, we calculate that there was a simultaneous depression of the ultrafiltration coefficient (Kf) during volume expansion with saline and possibly also to a lesser extent with albumin. The hypofiltration during either infusion delayed the onset of a natriuretic response until the filtered sodium load was restored to baseline in the postinfusion period. We propose that the net effect of changes in intracapillary pressures and Kf during volume expanding infusions is to transiently lower GFR, thereby preventing the human kidney from mounting an immediate natriuretic response to acute hypervolemia.

Adult↗