Biomedical subjects

William M Deen

Publications and source records attributed to William M Deen.

18 recordsLinked to original sources

PubMed2006

Model of albumin reabsorption in the proximal tubule.

Normally, the small amount of albumin which passes through the glomerular capillary wall is almost completely reabsorbed in the proximal tubule, via an endocytic mechanism, but the reabsorptive process can be overwhelmed if the filtered load of albumin is too large. To examine the factors that control the fractional reabsorption of albumin (f), we developed a mathematical model which assumes saturable endocytosis kinetics with a maximum reabsorptive capacity, V(max), and which includes the effects of flow and diffusion in the lumen. Limitations in albumin transport from the bulk tubule fluid to the endocytic sites at the bases of the microvilli had only a modest (8%) effect on the value of V(max) needed to fit micropuncture data on tubule albumin concentrations in rats. For moderate changes in filtered load, there was much greater sensitivity of f to SNGFR than to the albumin concentration of the filtrate (C(0)). A 50% increase in SNGFR was predicted to cause four- to fivefold increases in albumin excretion in rats or humans. For large increases in C(0), as might result from defects in glomerular sieving, there was a threshold at which the reabsorptive process became saturated and f fell sharply. That threshold corresponded to sieving coefficients of 10(-3) to 10(-2), the higher values occurring at reduced SNGFR. The predictions of the present model contrast with those of one proposed recently by Smithies (32), which does not include the effects of tubule flow rate.

Albumins↗

PubMed2005

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 degrees C were found to be (2.21+/-0.02) x 10(-5) cm2 s(-1) and (2.21+/-0.04) x 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 degrees C yielded a value of 3.0 x 10(-5) cm2 s(-1). The solubility of NO in water at 25 degrees C was (1.94+/-0.03) x 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 degrees C was (1.75+/-0.02) x 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.

Diffusion↗

PubMed2005

Nitric oxide, oxygen, and superoxide formation and consumption in macrophage cultures.

To examine the potential for generating toxic nitrogen oxides during the immune response, rates of formation and consumption of NO, O2, and O2- were measured in murine macrophage-like RAW264.7 cells. Cellular kinetic parameters for NO and O2 were obtained by monitoring their time-dependent concentrations in a closed chamber, and net cellular synthesis of O2- was quantified from ferricytochrome c reduction in cultures where NO synthesis was inhibited. Also measured was the photosensitive generation of O2- in the culture media. Unactivated cells (without NO synthesis) had an O2 consumption rate of 32+/-3 pmol s-1 (10(6) cells)-1, typical of mammalian cells. Also typical was that adding NO rapidly and reversibly inhibited respiration. Activated cells synthesized NO at a rate of 4.9+/-0.6 pmol s-1 (10(6) cells)-1. When NO synthesis was inhibited, they consumed three times as much O2 as unactivated cells [108+/-17 pmol s-1 (10(6) cells)-1]; however, O2 consumption of activated cells exposed to 1 microM NO was calculated to be comparable to that of NO-free unactivated cells. Rates of intracellular NO consumption were small, implying that enzymatic consumption does little to limit net NO synthesis by macrophages. Accounting for O2- generation in the culture media resulted in net rates of cellular O2- synthesis smaller than previously reported; the rate was 6% of NO synthesis in activated cells and was undetectable in unactivated cells.

Animals↗

PubMed2004

Hindered convection of macromolecules in hydrogels.

Hindered convection of macromolecules in gels was studied by measuring the sieving coefficient (theta) of narrow fractions of Ficoll (Stokes-Einstein radius, r(s) = 2.7-5.9 nm) in agarose and agarose-dextran membranes, along with the Darcy permeability (kappa). To provide a wide range of kappa, varying amounts of dextran (volume fractions < or = 0.011) were covalently attached to agarose gels with volume fractions of 0.040 or 0.080. As expected, theta decreased with increasing r(s) or with increasing concentrations of either agarose or dextran. For each molecular size, theta plotted as a function of kappa fell on a single curve for all gel compositions studied. The dependence of theta on kappa and r(s) was predicted well by a hydrodynamic theory based on flow normal to the axes of equally spaced, parallel fibers. Values of the convective hindrance factor (K(c), the ratio of solute to fluid velocity), calculated from Theta and previous equilibrium partitioning data, were unexpectedly large; although K(c) < or = 1.1 in the fiber theory, its apparent value ranged generally from 1.5 to 3. This seemingly anomalous result was explained on the basis of membrane heterogeneity. Convective hindrances in the synthetic gels were quite similar to those in glomerular basement membrane, when compared on the basis of similar solid volume fractions and values of kappa. Overall, the results suggest that convective hindrances can be predicted fairly well from a knowledge of kappa, even in synthetic or biological gels of complex composition.

Basement Membrane↗

PubMed2004

Equilibrium partitioning of Ficoll in composite hydrogels.

Equilibrium partition coefficients (phi, the concentration in the gel divided by that in free solution) of fluorescein-labeled Ficolls in pure agarose and agarose-dextran composite gels were measured as a function of gel composition and Ficoll size. The four narrow fractions of Ficoll, a spherical polysaccharide, had Stokes-Einstein radii ranging from 2.7 to 5.9 nm. Gels with agarose volume fractions of 0.040 and 0.080 were studied, with dextran volume fractions (calculated as if the chain were a long fiber) up to 0.011. As expected, phi generally decreased as the Ficoll size increased (for a given gel composition) or as the amount of dextran incorporated into the gel increased (for a given agarose concentration and Ficoll size). The decrease in phi that accompanied dextran addition was predicted well by an excluded volume theory in which agarose and dextran were both treated as rigid, straight, randomly positioned and oriented fibers. Modeling dextran as a spherical coil within a fibrous agarose gel produced much less accurate predictions. The diffusional permeabilities of these gels were assessed by combining the current partitioning data with relative diffusivities (Kd, the diffusivity in the gel divided by that in free solution) reported previously. The values of phi Kd for a synthetic gel with 8.0% agarose and 1.1% dextran (by volume) were found to be very similar to those for the glomerular basement membrane, a physiologically important material which also has a total solids content of approximately 10%.

Dextrans↗

PubMed2004

Effects of concentration on the partitioning of macromolecule mixtures in agarose gels.

To test the effects of solute concentration on the equilibrium partitioning of single macromolecules and macromolecule mixtures between bulk solutions and gels, the partition coefficient in agarose was measured for BSA and for four narrow fractions of Ficoll with Stokes radii of 30-59 A. Solutions of each test macromolecule were equilibrated with a known volume of gel, final liquid concentrations measured, and partition coefficients (gel concentration divided by bulk concentration) calculated by applying a material balance. The partition coefficient of each macromolecule was measured in 4 and 6% gels under dilute conditions and with BSA present at initial concentrations up to 13.5 g/dl. As expected, the partition coefficients decreased with increasing agarose concentration and with increasing macromolecular size. Moreover, increasing the BSA concentration increased the partition coefficient of BSA itself and that of all four Ficolls. This effect was most pronounced for the largest test solutes. Measurements at two ionic strengths confirmed that electrostatic interactions were negligible under the conditions used. The experimental results were compared with predictions from a previously developed excluded volume theory for the partitioning of mixtures of rigid, spheroidal macromolecules in fibrous media. Agarose was represented as a randomly oriented array of cylindrical fibers, BSA as a prolate spheroid, and Ficoll as a sphere. The quantitative agreement between the model predictions and the data was generally quite good, indicating that steric interactions among solute molecules and between solute molecules and gel fibers could explain the partitioning results. The theory is simple enough computationally to be applied to a variety of processes that are influenced by the equilibrium partitioning of macromolecules.

Animals↗

PubMed2004

Analysis of the effects of nitric oxide and oxygen on nitric oxide production by macrophages.

The interactions between NO and O(2) in activated macrophages were analysed by incorporating previous cell culture and enzyme kinetic results into a novel reaction-diffusion model for plate cultures. The kinetic factors considered were: (i) the effect of O(2) on NO production by inducible NO synthase (iNOS); (ii) the effect of NO on NO synthesis by iNOS; (iii) the effect of NO on respiratory and other O(2) consumption; and (iv) the effects of NO and O(2) on NO consumption by a possible NO dioxygenase (NOD). Published data obtained by varying the liquid depth in macrophage cultures provided a revealing test of the model, because varying the depth should perturb both the O(2) and the NO concentrations at the level of the cells. The model predicted that the rate of NO(2)(-) production should be nearly constant, and that the net rate of NO production should decline sharply with increases in liquid depth, in excellent agreement with the experimental findings. In further agreement with available results for macrophage cultures, the model predicted that net NO synthesis should be more sensitive to liquid depth than to the O(2) concentration in the headspace. The main reason for the decrease in NO production with increasing liquid depth was the modulation of NO synthesis by NO, with O(2) availability playing only a minor role. The model suggests that it is the ability of iNOS to consume NO, as well as to synthesize it, that creates very sensitive feedback control, setting an upper bound on the NO concentration of approximately 1 microM. The effect of NO consumption by other possible pathways (e.g., NOD) would be similar to that of iNOS, in that it would help limit net NO production. The O(2) utilized during enzymatic NO consumption is predicted to make the O(2) demands of activated macrophages much larger than those of unactivated ones (where iNOS is absent); this remains to be tested experimentally.

Animals↗

PubMed2004

Peroxynitrite delivery methods for toxicity studies.

The endogenous synthesis of peroxynitrite (ONOO(-)) has been implicated in a number of diseases, but assessments of its cytotoxicity and genotoxicity have been hampered by its extremely short half-life under physiological conditions (<20 ms) and the consequent difficulty in exposing cells to known concentrations of it over at least several hours. Two methods for peroxynitrite delivery to cell cultures were investigated, one involving steady infusion of preformed ONOO(-) and the other based on the continuous in situ synthesis of ONOO(-) from NO and O(2)(-). In the latter, NO was supplied by diffusion through gas permeable tubing and O(2)(-) was generated using the hypoxanthine-xanthine oxidase reaction. The performance of both methods was assessed by measuring the rates of formation of tyrosine derivatives (dityrosine and nitrotyrosine) that are commonly employed as biomarkers for peroxynitrite. Experimental results in the absence of cells were compared in each case with predictions from kinetic models. In the infusion system, the measured dityrosine and nitrotyrosine yields were in excellent agreement with those predicted from the model. To characterize the other system, experiments were performed first to determine the kinetics of hypoxanthine oxidation by xanthine oxidase and uric acid oxidation by uricase. Simulations of the complex reaction network in the complete synthesis system suggested that dityrosine should be the major product there, that the yields of both tyrosine derivatives should be very sensitive to the relative rates of NO and O(2)(-) delivery, and that equal rates for NO and O(2)(-) should maximize those yields. Experiments performed under the predicted optimal conditions showed much lower levels of dityrosine than expected and no detectable nitrotyrosine. The unexpectedly low yields of tyrosine products could be explained largely by the partial inactivation of both xanthine oxidase and uricase by peroxynitrite-derived NO(2) and CO(3)(-) radicals. We conclude that continuous infusion of peroxynitrite is the more promising approach.

Animals↗

PubMed2004

Peroxynitrite exposure of cells cocultured with macrophages.

Peroxynitrite is a potent oxidant that is formed endogenously from NO and O2-. One approach for studying its toxicity involves the coculture of activated macrophages (producers of NO and O2-) with "target cells" (which make O2- but not NO). Because peroxynitrite concentrations in such experiments are too small and localized to measure, reaction-diffusion models were developed for situations ranging from isolated cells to many cells randomly distributed on a plate. In these models the macrophage population created a constant NO concentration in and near all cells, whereas fluxes of O2- into the media from individual macrophages led to localized extracellular formation of peroxynitrite. Target cells were found to have no effect on the peroxynitrite concentrations in other cells, whereas the macrophage effects were additive. The average concentration inside randomly distributed target cells increased with increasing macrophage number density, as entry of extracellular peroxynitrite grew in importance relative to intracellular formation. For high cell densities, large peroxynitrite membrane permeabilities, and low rates of intracellular synthesis, the surrounding macrophages were calculated to double or triple the peroxynitrite concentration in an average target cell. It was estimated that a macrophage enveloping a target cell could cause as much as a 10-fold increase in the concentration inside that target cell.

Animals↗

PubMed2004

Glomerular filtration of albumin: how small is the sieving coefficient?

A model was developed to describe how the concentration of albumin in proximal tubule fluid will vary with axial position, including the effects of luminal flow and water and albumin reabsorption. The results show that the high albumin sieving coefficients proposed recently cannot be reconciled with micropuncture data in rats.

Albuminuria↗

PubMed2004

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.

Animals↗

PubMed2003

Analysis of cellular exposure to peroxynitrite in suspension cultures.

A mathematical model was developed to predict the intracellular concentrations of NO, O(2)(-), and peroxynitrite in suspension cell cultures exposed to NO and/or peroxynitrite. Oxygen and CO(2) were also considered. Steady state concentrations were computed as a function of radial position within an idealized spherical cell, with a distinction being made between cytosolic and mitochondrial values. Spatial variations in the intracellular concentrations of O(2), CO(2), and NO were found to be negligible. The extremely low membrane permeabilities for O(2)(-) (estimated from lipid bilayer data) caused O(2)(-) to be consumed in the compartment in which it was generated (mitochondria or cytosol) and resulted in concentrations that depended on the generation rate and the concentrations of superoxide dismutase and NO in the individual compartments. Special attention was paid to the origins of intracellular peroxynitrite. Potential sources of peroxynitrite include intracellular generation in mitochondria and cytosol and (depending on the type of experiment) diffusion of extracellular peroxynitrite into the cell. The relative importance of extracellular and intracellular sources was estimated for a wide variety of conditions. The calculated mitochondrial concentrations were generally 5-10 times higher than the cytosolic values, and it was found that mitochondria may act either as sources or sinks for cytosolic peroxynitrite, depending on the experimental conditions. For the baseline conditions, including an NO concentration of 1 microM and no peroxynitrite in the medium, the cytosolic peroxynitrite concentration was estimated as approximately 2 nM. The extracellular peroxynitrite concentration required to double the cytosolic level was approximately 25 nM, and an extracellular concentration of approximately 100 nM was needed to effect a 5-fold increase. For extracellular concentrations smaller than 25 nM, intracellular generation predominated.

Animals↗

PubMed2003

Thresholds of nitric oxide-mediated toxicity in human lymphoblastoid cells.

A novel delivery system was used to study NO-mediated cyto- and genotoxicity in two human lymphoblastoid cell lines, TK6 (wild-type p53) and NH32 (p53-null but isogenic to TK6). The delivery system, which supplied NO and O(2) continuously by diffusion through gas permeable tubing, was found to maintain the NO and O(2) concentrations at constant, predictable values. Cellular rates of NO and O(2) consumption and mass transfer coefficients for the two gases were measured in separate experiments and used to calculate the NO concentrations during exposure experiments. The TK6 and NH32 cells were each exposed to several steady state NO concentrations for varying lengths of time, so that the total dose (area under the concentration-time curve) covered a wide range. End point assays, including lethality, apoptosis, mitochondrial damage, and mutation rate in the thymidine kinase (TK1) gene locus, were performed at different posttreatment times. Control experiments using Ar instead of NO resulted in normal cell proliferation for all exposure times tested (up to 36 h). As compared to those controls, significant cell death, apoptosis, and mitochondrial membrane depolarization were observed in NO-treated TK6 cells, and the TK1 mutation rate was elevated. Of particular importance, toxic effects were observed only when the NO concentration and dose were greater than threshold values of approximately 0.5 micro M and approximately 150 micro M min, respectively. If neither or only one threshold was exceeded, the effects were insignificant; when both were exceeded, total cell survival and the number of nonapoptotic cells both decreased exponentially with increasing NO dose. In general, the NH32 cells were much more resistant to NO-induced damage and death than TK6 cells, demonstrating that p53 status is an important determinant of NO-induced cytotoxicity.

Apoptosis↗

PubMed2003

Absence of 2'-deoxyoxanosine and presence of abasic sites in DNA exposed to nitric oxide at controlled physiological concentrations.

Nitric oxide (NO(*)) is a physiologically important molecule at low concentrations, while high levels have been implicated in the pathophysiology of diseases associated with chronic inflammation, such as cancer. While an extensive study in vitro suggests that oxidative and nitrosative reactions dominate the complicated chemistry of NO(*)-mediated genotoxicity, neither the spectrum of DNA lesions nor their consequences in vivo have been rigorously defined. We have approached this problem with a major effort to define the spectrum of nitrosative DNA lesions produced by NO(*)-derived reactive nitrogen species under biological conditions. Plasmid pUC19 DNA was exposed to steady state concentrations of 1.3 microM NO(*) and 190 microM O(2) (calculated steady state concentrations of 40 fM N(2)O(3) and 3 pM NO(2)(*) in the bulk solution) in a recently developed reactor that avoids the undesired gas phase chemistry of NO(*) and approximates the conditions at sites of inflammation in tissues. The resulting spectrum of nitrosatively induced abasic sites and nucleobase deamination products was defined using plasmid topoisomer analysis and a novel LC/MS assay, respectively. With a limit of detection of 100 fmol and a sensitivity of 6 lesions per 10(7) nt in 50 microg of DNA, the LC/MS analysis revealed that 2'-deoxyxanthosine (dX), 2'-deoxyinosine (dI), and 2'-deoxyuridine (dU) were formed at nearly identical rates (k = 1.2 x 10(5) M(-1) s(-1)) to the extent of approximately 80 lesions per 10(6) nt after 12 h exposure to NO(*) in the reactor. While reactions with HNO(2) resulted in the formation of high levels of 2'-deoxyoxanosine (dO), one of two products arising from deamination of dG, dO, was not detected in 500 microg of DNA exposed to NO(*) in the reactor for up to 24 h (<6 lesions per 10(8) nt). This result leads to the prediction that dO will not be present at significant levels in inflamed tissues. Another important observation was the NO(*)-induced production of abasic sites, which likely arise by nitrosative depurination reactions, to the extent of approximately 10 per 10(6) nt after 12 h of exposure to NO(*) in the reactor. In conjunction with other studies of nitrosatively induced dG-dG cross-links, these results lead to the prediction of the following spectrum of nitrosative DNA lesions in inflamed tissues: approximately 2% dG-dG cross-links, 4-6% abasic sites, and 25-35% each of dX, dI, and dU.

DNA Damage↗

PubMed2003

Nitric oxide delivery system for cell culture studies.

To investigate the toxicity and mutagenicity of NO, methods are needed to deliver it to cell cultures at known, constant rates. To permit continuous exposures over lengthy periods, we fabricated a simple apparatus utilizing gas-permeable polydimethylsiloxane (Silastic) tubing to supply both NO and O2 to a stirred, cylindrical vessel. Mass transfer in this system was characterized by measuring the delivery rates of NO or O2 alone, and of NO to air-saturated solutions. The concentrations of NO, O2, and NO2- (the end product of NO oxidation) were monitored continuously. The total flux of nitrogen species into the liquid (as determined from the sum of NO and NO2- accumulation) was 50%-90% greater in the presence of O2, depending on the NO partial pressure in the gas. Also, the simultaneously measured mass transfer coefficients for NO and O2 differed greatly from the corresponding unreactive values. An analysis of the data using diffusion-reaction models showed that NO oxidation in the aqueous boundary layer contributed very little to the nitrogen flux increase or to variations in the mass transfer coefficients. However, the unusually strong dependence of the delivery rates on chemical reactions could be explained by postulating that partial oxidation of NO to NO2 occurred within the membrane. The rate constant we estimated for polydimethylsiloxane, 4.4 x 10(5) M-2 s(-1) at 23 degrees C, is only about one-fifth of values reported previously for water and nonpolar solvents, but the high solubilities of NO and O2 in the polymer are sufficient to make NO2 formation significant. Although considerable NO2 is calculated to enter the liquid, its reaction with aqueous NO is rapid enough to keep this undesired compound at trace levels, except within a few microns of the tubing. Thus, cells will have little exposure to NO2

Cell Culture Techniques↗

PubMed2002

Agarose-dextran gels as synthetic analogs of glomerular basement membrane: water permeability.

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(a)) of 0.04 or 0.08 and dextran volume fractions (phi(d)) 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 kappa for pure agarose were obtained for remarkably small volume fractions of dextran: phi(d) > or = 0.0003 for phi(a) = 0.04 and phi(d) > or = 0.001 for phi(a) = 0.08. The Darcy permeabilities were much more sensitive to phi(d) than to phi(a), and were as much as 26 times smaller than those of pure agarose. Although phi(d) was an important variable, dextran molecular weight was not. The effects of dextran addition on kappa 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 nm2) makes them an attractive experimental model for glomerular basement membrane and possibly other extracellular matrices.

Biophysical Phenomena↗

PubMed2002

Effect of liquid depth on the synthesis and oxidation of nitric oxide in macrophage cultures.

The effect of liquid depth on the synthesis of NO and O(2)(-) was studied in murine macrophage-like RAW 264.7 cells activated by bacterial lipopolysaccharide and interferon-gamma. Rates of NO(2)(-) and NO(3)(-) accumulation were determined 8-11 h after stimulation. The rate of NO synthesis was computed by using a reaction-diffusion model to correct NO(2)(-) and NO(3)(-) accumulation for physical loss of NO, whereas O(2)(-) synthesis was equated with NO(3)(-) formation. Rates of O(2)(-) synthesis determined by a spectrophotometric (cytochrome c) assay were in good agreement with those from NO(3)(-) accumulation and showed production of O(2)(-) to be detectable immediately, in contrast to the approximately 6 h time lag for NO. The assumption that NO(2)(-) and NO(3)(-) are stable end products of the extracellular oxidation of NO by O(2) and O(2)(-), respectively, was supported by the fact that NO(2)(-) and NO(3)(-) concentrations remained constant in the presence of unstimulated cells or stimulated cells where NO synthesis was inhibited. Data were obtained for media depths ranging from 1 to 4 mm. The physical loss of NO was found to be quite significant, exceeding NO(2)(-) and NO(3)(-) accumulation by an order of magnitude at the smallest depth. The principal finding was that the rates of NO(2)(-) and NO(3)(-) accumulation each remained nearly constant over the 4-fold range of liquid depths. Because greater depths should greatly facilitate the trapping of NO as NO(2)(-), this implies that NO synthesis decreased markedly with increasing depth. In contrast, O(2)(-) synthesis remained approximately constant. Oxygen availability is likely to have affected NO synthesis, in that diffusional limitations will yield the lowest cellular O(2) concentrations when the liquid depth is greatest and NO synthesis is known to decrease when O(2) levels are reduced. Concentrations of NO near the cells were calculated to remain at approximately 1 microM for all conditions examined, suggesting that regulation of NO synthase activity by NO might also have mediated the effect of liquid depth.

Animals↗

PubMed2002

Maintenance and recovery stages of postischemic acute renal failure in humans.

Postischemic injury in 38 recipients of 7-day-old cadaveric renal allografts was classified into sustained (n = 15) or recovering (n = 23) acute renal failure (ARF) according to the prevailing inulin clearance. Recipients of long-standing allografts that functioned optimally (n = 16) and living transplant donors undergoing nephrectomy (n = 10) served as functional and structural controls, respectively. A combination of physiological and morphometric techniques were used to evaluate glomerular filtration rate and its determinants 1-3 h after reperfusion and again on day 7 to elucidate the mechanism for persistent hypofiltration in ARF that is sustained. Glomerular filtration rate in the sustained ARF group on day 7 was depressed by 90% (mean +/- SD); the corresponding fall in renal plasma flow was proportionately less. Neither plasma oncotic pressure nor the single-nephron ultrafiltration coefficient differed between the sustained ARF and the control group, however. A model of glomerular ultrafiltration and a sensitivity analysis were used to compute the prevailing transcapillary hydraulic pressure gradient (DeltaP), the only remaining determinant of DeltaP. This revealed that DeltaP varied between 27 and 28 mmHg in sustained ARF and 32-38 mmHg in recovering ARF on day 7 vs. 47-54 mmHg in controls. Sustained ARF was associated with persistent tubular dilatation. We conclude that depression of DeltaP, perhaps due partially to elevated tubule pressure, is the predominant cause of hypofiltration in the maintenance stage of ARF that is sustained for 7 days.

Acute Kidney Injury↗