Analysis of standing droplets in rat proximal tubules

Volume, osmolality, and concentrations for Na, Cl, and raffinose have been measured as a function of time in standing droplets within rat intermediate and late proximal tubules. Standing droplet reabsorption proceeds without the development of a measurable osmotic difference across the epithelium. After 140 s of tubular exposure, droplet-to- plasma concentration differences are observed for raffinose, Na, and Cl with the observed Na concentration difference, usually referred to as limiting gradient, being approximately 9 mM. It is possible that a smaller or even no limiting difference would be attained with longer exposure times. Previous values measured for the limiting Na concentration in the rat proximal tubule were determined before the attainment of constant concentrations. Assuming that the Na concentration we measured is the limiting value, we estimate that active NaCl transport accounts for a very small fraction, less than 6%, of the volume reabsorption; using an alternative approach of fitting a theoretical model to our experimental data, active NaCl transport is again estimated to account for only 6% of the total reabsorbate. The previous interpretation that a limiting Na concentration gradient constitutes the most direct evidence for active Na transport may be in error; the gradient we measure can be modeled without incorporating active NaCl transport.     

Epithelial water transport in a balanced gradient system.

The relationship between epithelial fluid transport, standing osmotic gradients, and standing hydrostatic pressure gradients has been investigated using a perturbation expansion of the governing equations. The assumptions used in the expansion are: (a) the volume of lateral intercellular space per unit volume of epithelium is small; (b) the membrane osmotic permeability is much larger than the solute permeability. We find that the rate of fluid reabsorption is set by the rate of active solute transport across lateral membranes. The fluid that crosses the lateral membranes and enters the intercellular cleft is driven longitudinally by small gradients in hydrostatic pressure. The small hydrostatic pressure in the intercellular space is capable of causing significant transmembrane fluid movement, however, the transmembrane effect is countered by the presence of a small standing osmotic gradient. Longitudinal hydrostatic and osmotic gradients balance such that their combined effect on transmembrane fluid flow is zero, whereas longitudinal flow is driven by the hydrostatic gradient. Because of this balance, standing gradients within intercellular clefts are effectively uncoupled from the rate of fluid reabsorption, which is driven by small, localized osmotic gradients within the cells. Water enters the cells across apical membranes and leaves across the lateral intercellular membranes. Fluid that enters the intercellular clefts can, in principle, exit either the basal end or be secreted from the apical end through tight junctions. Fluid flow through tight junctions is shown to depend on a dimensionless parameter, which scales the resistance to solute flow of the entire cleft relative to that of the junction. Estimates of the value of this parameter suggest that an electrically leaky epithelium may be effectively a tight epithelium in regard to fluid flow.     

Isosmotic volume reabsorption in rat proximal tubule

A theoretical model incorporation both active and passive forces has been developed for fluid reabsorption from split oil droplets in rat intermediate and late proximal tubule. Of necessity, simplifying assumptions have been introduced; we have assumed that the epithelium can be treated as a single membrane and that the membrane "effective" HCO3 permeability is near zero. Based on this model with its underlying assumptions, the following conclusions are drawn. Regardless of the presence or absence of active NaCl transport, fluid reabsorption from the split oil droplet is isosmotic. The reabsorbate osmolarity can be affected by changes in tubular permeability parameters and applied forces but is not readily altered from an osmolarity essentially equal to that of plasma. In a split droplet, isosmotic flow need not be a special consequence of active Na transport, is not the result of a particular set of permeability properties, and is not merely a trivial consequence of a very high hydraulic conductivity; isosmotic flow can be obtained with hydraulic conductivity nearly an order of magnitude lower than that previously measured in the rat proximal convoluted tubule. Isosmotic reabsorption is, in part, the result of the interdependence of salt and water flows, their changing in parallel, and thus their ratio, the reabsorbate concentration being relatively invariant. Active NaCl transport can cause osmotic water flow by reducing the luminal fluid osmolarity. In the presence of passive forces the luminal fluid can be hypertonic to plasma, and active NaCl transport can still exert its osmotic effect on volume flow. There are two passive forces for volume flow: the Cl gradient and the difference in effective osmotic pressure; they have an approximately equivalent effect on volume flow. Experimentally, we have measured volume changes in a droplet made hyperosmotic by the addition of 50 mM NaCl; the experimental results are predicted reasonably well by our theoretical model.     

Application of a fiber-matrix model to transport in renal tubules

The effects of tight junction structure on water and solute fluxes across proximal tubular epithelium were examined with fiber-matrix equations previously derived by Curry and Michel (1980. Microvascular Research. 20:96-99). Using plausible estimates of tight junction fiber length and width the model predicts solute (Ps) and water permeability (Lp) coefficients that agree with the measured values. When fiber-matrix and pore models were compared for physiologically relevant ranges of matrix void fraction (80-98%) and pore radii (0-20 A), the fiber-matrix model predicted a 10-fold higher Lp/Ps ratio. Lp/Ps was most sensitive to small changes in tight junction structure when void fractions exceeded 90%. Void fractions of 96.5% and 97.1% predicted previously measured values for Lp and solute permeabilities in rat and rabbit proximal tubules. These values are consistent with void fractions and permeabilities of artificial membranes. The fiber-matrix tight junction model was incorporated into a model of reabsorption from the rat proximal tubule developed by Weinstein (1984). American Journal of Physiology. 247:F848-F862.) A void fraction of 98% predicted the experimental results for isosmotic reabsorption driven by active transport. Changing void fraction over the range of 97-99% produced a 50-75% change in predicted volume reabsorption with active transport. According to the fiber-matrix model: (a) solute permeabilities alone cannot be used to predict Lp, (b) previously measured solute permeabilities in the proximal tubule are compatible with significant water reabsorption through a water-permeable tight junction, and (c) hydraulic and solute permeabilities may be sensitive to small changes in tight junction fiber length and diameter or ionic strength within the tight junction.     

Transepithelial sulfate transport by avian renal proximal tubule epithelium in primary culture

The mechanisms and control of transepithelial inorganic sulfate (Si) transport by primary cultures of chick renal proximal tubule monolayers in Ussing chambers were determined. The competitive anion, S2 O 3 2- (5 mM), reduced both unidirectional reabsorptive and secretory fluxes and net Si reabsorption with no effect on electrophysiological properties. The carbonic anhydrase (CA) inhibitor ethoxzolamide decreased net Si reabsorption approximately 45%. CAII protein and activity were detected in isolated chick proximal tubules by immunoblots and biochemical assay, respectively. Cortisol reduced net Si reabsorption up to approximately 50% in a concentration-dependent manner. Thyroid hormone increased net Si reabsorption threefold in 24 h, and parathyroid hormone (PTH) acutely stimulated net Si reabsorption approximately 45%. These data indicate that CA participates in avian proximal tubule active transepithelial Si reabsorption, which cortisol directly inhibits and T3 and PTH directly stimulate.     

Mechanism and Control of Fluid Secretion

Fluid secretion and reabsorption by a variety of plant and animal tissues appear to be accomplished by osmotic coupling between solute transport and water movement. The local osmosis model suggests that active accumulation of solutes within narrow folds at the cell surface may produce the local gradients that generate water flow. Both micropuncture techniques and electron-probe X-ray microanalysis have established that local osmotic gradients occur in absorptive epithelia, but they have not as yet been detected in secretory tissues.Hormonal control of secretion involves stimulation of solute pumps and adjustments of permeability to non-transported solutes. Since hormone receptors and pumps are often located on opposite surfaces of the cell, intracellular second messengers convey the secretory signal through cytoplasm. Much has been learned by study of insect tissues that are anatomically simple and that function for long periods in vitro. Aspects of hormone-receptor interaction have been explored, including the action of halluninogenic molecules. In insect salivary glands cyclic AMP appears to stimulate cation transport, while calcium increases anion permeability. The various second messengers probably interact with each other in complex feedback loops that stabilize the system and make it quickly responsive to hormone. Cyclic AMP may stimulate release of calcium from mitochondria. Unresolved is the way second messengers alter properties of the cell surface.     

Osmotic Transport across Cell Membranes in Nondilute Solutions: A New Nondilute Solute Transport Equation

The fundamental physical mechanisms of water and solute transport across cell membranes have long been studied in the field of cell membrane biophysics. Cryobiology is a discipline that requires an understanding of osmotic transport across cell membranes under nondilute solution conditions, yet many of the currently-used transport formalisms make limiting dilute solution assumptions. While dilute solution assumptions are often appropriate under physiological conditions, they are rarely appropriate in cryobiology. The first objective of this article is to review commonly-used transport equations, and the explicit and implicit assumptions made when using the two-parameter and the Kedem-Katchalsky formalisms. The second objective of this article is to describe a set of transport equations that do not make the previous dilute solution or near-equilibrium assumptions. Specifically, a new nondilute solute transport equation is presented. Such nondilute equations are applicable to many fields including cryobiology where dilute solution conditions are not often met. An illustrative example is provided. Utilizing suitable transport equations that fit for two permeability coefficients, fits were as good as with the previous three-parameter model (which includes the reflection coefficient, σ). There is less unexpected concentration dependence with the nondilute transport equations, suggesting that some of the unexpected concentration dependence of permeability is due to the use of inappropriate transport equations.     

Axial heterogeneity and filtered-load dependence of proximal bicarbonate reabsorption.

A theoretical model was developed to examine the role of physical and chemical factors in the control of bicarbonate reabsorption in the renal proximal tubule. Included in the model were axial and radial variations in the concentrations of HCO3-, CO2 and related chemical species in the tubule lumen and epithelial cells. Relations between these concentrations and the solute fluxes across the brush border and basolateral membranes were also included, as were reaction rate and equilibrium expressions to describe the various buffering processes in the lumen and cells. The two most critical membrane parameters, the rate constant for H+ secretion at the brush border and the effective permeability of HCO3- at the basolateral membrane, were evaluated by comparing model predictions with available free-flow micropuncture data in the rat. It was found that the experimental observations could be explained only by decreasing one or both of these membrane parameters with axial position, suggesting a progressive decrease in HCO3- reabsorptive capacity along the tubule. For single nephron filtered loads of HCO3- up to about 1,400 pmol/min, absolute bicarbonate reabsorption was predicted to increase nearly in proportion to filtered load, whereas it was calculated to be relatively constant at higher filtered loads, irrespective of how filtered load was assumed to be varied. These predictions are in excellent agreement with most of the available micropuncture data in rats, as is the prediction that HCO3- reabsorption should change in parallel with CO2 partial pressure in the filtrate, at a given filtered load of HCO3-. Certain discrepancies between the model predictions and experimental observations are evident at very high filtered loads, and the implications of these are discussed in terms of possible adaptive responses of the tubule.     

Structural organization of nephrons in the kideny and proximal reabsorption in representatives of different vertebrate classes (according to microdissection and micropuncture studies)]

By microdissection method, single nephrons have been isolated from the river lamprey Lampetra fluviatilis, teleost fish Myoxocephalus scorpius (White sea), frog Rana temporaria, lizard Agama caucasica, hen Gallus domesticus and albino rat Rattus norvegicus (Wistar strain). Tubular reabsorptive capacity was measured in kidneys of lampreys and rats by the split oil droplet method. In the animals studied, each of the nephrons consists of the glomerulus, proximal, thin and distal segments. Relative length of the proximal segment does not depend on phylogenetic position of a species. Transtubular isotonic water reabsorption from the proximal tubule in rats is significantly higher than in lampreys. The level of proximal reabsorption is not related to the length of the proximal tubule in the Verterbrates studied but depends on the intensity of tubular transport. The structure which is similar to Henle's loop is present already in Cyclostomes [12]. Further stage of the progressive development of the uriniferous tubules is presented by uneven localization of nephron populations within the kideney in Reptiles. These data suggest that the effective system of osmotic concentration appeared in evolution not as the result of Henle's loop formation, but on the basis of the development of the medullar substance formed mainly by intercortical and juxtamedullar nephron populations.     

Convective paracellular solute flux. A source of ion-ion interaction in the epithelial transport equations

An electrolyte model of an epithelium (a cell and a tight junction in parallel, both in series with a lateral interspace basement membrane) is analyzed using the formalism of nonequilibrium thermodynamics. It is shown that if the parallel structures are heteroporous (i.e., reflection coefficients for two ion species differ between the components), then a cross-term will appear in the overall transport equations of the epithelium. Formally, this cross-term represents an ion-ion interaction. With respect to the rat proximal tubule, data indicating epithelial ionic reflection coefficients less than unity, together with the assumption of no transcellular solvent drag, imply the presence of convective paracellular solute flux. This means that a model applicable to a heteroporous structure must be used to represent the tubule, and, in particular, the cross-terms for ion-ion interaction must also be evaluated in permeability determinations. A series of calculations is presented that permits the estimation of the Na-Cl interaction for rat proximal tubule from available experimental data. One consequence of tubule heteroporosity is that an electrical potential may be substantially less effective than an equivalent concentration gradient in driving reabsorptive ion fluxes.     

Coupled water transport in standing gradient models of the lateral intercellular space.

A standing gradient model of the lateral intercellular space is presented which includes a basement membrane of finite solute permeability. The solution to the model equations is estimated analytically using the "isotonic convection approximation" of Segel. In the case of solute pumps uniformly distributed along the length of the channel, the achievement of isotonic transport depends only on the water permeability of the cell membranes. The ability of the model to transport water against an adverse osmotic gradient is the sum of two terms: The first term is simply that for a well-stirred compartment model and reflects basement membrane solute permeability. The second term measures the added strength due to diffusion limitation within the interspace. It is observed, however, that the ability for uphill water transport due to diffusion limitation is diminished by high cell membrane water permeability. For physiologically relevant parameters, it appears that the high water permeability required for isotonic transport renders the contribution of the standing gradient relatively ineffective in transport against an osmotic gradient. Finally, when the model transports both isotonically and against a gradient, it is shown that substantial intraepithelial solute polarization effects are unavoidable. Thus, the measured epithelial water permeability will grossly underestimate the water permeability of the cell membranes. The accuracy of the analytic approximation is demonstrated by numerical solution of the complete model equations.     

Comparison of the natriuresis and chlorures is associated with glomerular hyperfiltration induced by atrial natriuretic factor or glucagon

The impact on renal sodium chloride reabsorption of an acute increase in glomerular filtration rate (GFR) induced by atrial natriuretic factor (ANF) or glucagon was examined in the conscious rat. These hormones have no direct effect on proximal solute transport and have opposite effects on distal transport. ANF and glucagon increased GFR to a comparable extent (2.0 ± 0.2 to 3.5 ± 0.4 ml/min, p<0.01, and 1.9 ± 0.1 to 3.3 ± 0.1 ml/min, p<0.001, respectively). While most (95–97%) of the increment in filtered sodium chloride was reabsorbed, a small portion (3–5%) escaped tubular reabsorption. Absolute sodium and chloride urinary excretion rates increased similarly in response to each hormone, by two- to three-fold. Slightly imperfect load-dependent sodium chloride reabsorptive response by the nephron, despite opposite direct effects on distal nephron transport, may account for the observed natriuresis and chloruresis associated with the acute glomerular hyperfiltration induced by ANF or glucagon administration.     

A Biomechanical Triphasic Approach to the Transport of Nondilute Solutions in Articular Cartilage

Biomechanical models for biological tissues such as articular cartilage generally contain an ideal, dilute solution assumption. In this article, a biomechanical triphasic model of cartilage is described that includes nondilute treatment of concentrated solutions such as those applied in vitrification of biological tissues. The chemical potential equations of the triphasic model are modified and the transport equations are adjusted for the volume fraction and frictional coefficients of the solutes that are not negligible in such solutions. Four transport parameters, i.e., water permeability, solute permeability, diffusion coefficient of solute in solvent within the cartilage, and the cartilage stiffness modulus, are defined as four degrees of freedom for the model. Water and solute transport in cartilage were simulated using the model and predictions of average concentration increase and cartilage weight were fit to experimental data to obtain the values of the four transport parameters. As far as we know, this is the first study to formulate the solvent and solute transport equations of nondilute solutions in the cartilage matrix. It is shown that the values obtained for the transport parameters are within the ranges reported in the available literature, which confirms the proposed model approach.     

Role of the Paracellular Pathway in Isotonic Fluid Movement Across the Renal Tubule

Evidence for a highly permeable paracellular shunt in the proximal tubule is reviewed. The paracellular pathway is described as a crucial site for the regulation of net absorption and for solute-solvent interaction. Available models for the coupling of salt and water transport are assessed with respect to the problem of isotonic water movement. Two new models are proposed taking into account that the tight junctions are permeable to salt and water and that active transport sites for sodium are distributed uniformly along the lateral cell membrane. The first model (continuous model) is a modification of Diamond and Bossert''s proposal using different assumptions and boundary conditions. No appreciable standing gradients are predicted by this model. The second model (compartmental model) is an expansion of Curran''s double membrane model by including additional compartments and driving forces. Both models predict a reabsorbate which is not isosmotic. For the particular case of the proximal tubule it is shown that in the presence of a leaky epithelium these deviations from isotonicity might have escaped experimental observation.     

Sodium Movement across Single Perfused Proximal Tubules of Rat Kidneys

Using perfusion techniques in single proximal tubule segments of rat kidney, the relationship between net sodium movement and active transport of ions, as measured by the short-circuit method, has been studied. In addition, the role of the colloid-osmotic pressure gradient in proximal transtubular fluid and sodium movement has been considered. Furthermore, the limiting concentration gradient against which sodium movement can occur and the relationship between intratubular sodium concentration and fluid transfer have been investigated. Comparison of the short-circuit current with the reabsorptive movement of sodium ions indicates that this process is largely, perhaps exclusively, active in nature. No measurable contribution of the normally existing colloid-osmotic pressure gradient to transtubular water movement was detected. On the other hand, fluid movement across the proximal tubular epithelium is dependent upon the transtubular sodium gradient and is abolished when a mean concentration difference of 50 mEq/liter is exceeded.     

Identification of the site of glucocorticoid action on neutral amino acid transport in superficial nephrons of rat kidney

Summary. Glucocorticoid hormones enhance the reabsorptive capacity of filtered amino acids in rat kidney, as it was shown in previous in vivo clearance experiments. In the present study, the site of glucocorticoid action on neutral amino acid transport in superficial nephrons of rat kidney was investigated using in vivo micropuncture technique. Adult female Wistar rats were treated with dexamethasone (DEX), and fractional excretion of L-glutamine (L-Gln) and L-leucine (L-Leu) were determined and related to inulin after microinfusion into different nephron segments. DEX reduced fractional excretion of both neutral amino acids as a sign of enhanced reabsorptive capacity. The site of main DEX action on L-Leu reabsorption has been localized in the proximal straight tubule. However, in the case of L-Gln, the inhibition of γ-glutamyltranspeptidase (γ-GT) by administration of acivicin indicated the importance of this brush border enzyme in reduced L-Gln excretion. DEX enhanced γ-GT activity by tubular acidification. It can be presumed a DEX-inducible transport system for neutral amino acids mainly localized in proximal straight tubules of rat kidney. Received July 8, 1999     

Further Observations on Asymmetrical Solute Movement across Membranes

The permeability of frog skin under the influence of urea hyperosmolarity has been studied. Flux ratio asymmetry has been demonstrated again for tracer mannitol. The inhibitors DNP, CN^-, and ouabain have been used to eliminate active sodium transport and it was found that urea hyperosmolarity produces asymmetrical mannitol fluxes on frog skins having no short-circuit current. These findings suggest that flux ratio asymmetry is due to solute interaction and is unrelated to sodium transport. Studies with a synthetic membrane show clearly that bulk flow of fluid can produce a "solvent drag" effect and change flux ratios. When bulk flow is blocked and solute gradients allowed their full expression, then solute interaction "solute drag" is easily demonstrable in a synthetic system.     

Standing-gradient osmotic flow. A mechanism for coupling of water and solute transport in epithelia

At the ultrastructural level, epithelia performing solute-linked water transport possess long, narrow channels open at one end and closed at the other, which may constitute the fluid transport route (e.g., lateral intercellular spaces, basal infoldings, intracellular canaliculi, and brush-border microvilli). Active solute transport into such folded structures would establish standing osmotic gradients, causing a progressive approach to osmotic equilibrium along the channel's length. The behavior of a simple standing-gradient flow system has therefore been analyzed mathematically because of its potential physiological significance. The osmolarity of the fluid emerging from the channel's open end depends upon five parameters: channel length, radius, and water permeability, and solute transport rate and diffusion coefficient. For ranges of values of these parameters encountered experimentally in epithelia, the emergent osmolarity is found by calculation to range from isotonic to a few times isotonic; i.e., the range encountered in epithelial absorbates and secretions. The transported fluid becomes more isotonic as channel radius or solute diffusion coefficient is decreased, or as channel length or water permeability is increased. Given appropriate parameters, a standing-gradient system can yield hypertonic fluids whose osmolarities are virtually independent of transport rate over a wide range, as in distal tubule and avian salt gland. The results suggest that water-to-solute coupling in epithelia is due to the ultrastructural geometry of the transport route.     

Hyperoncotic albumin infusion in experimental glomerulonephritis in rats: A micropuncture study.

The effect of infusion of hyperonciotic (25 g% salt-poor bovine plasma albumin) (0.06 g% of body weight) has been studied using clearance and micropuncture techniques in animals with two forms of immunologically induced experimental glomerulonephritis and in age-matched control rats. In control rats the significant natriuresis and diuresis after hyperoncotic albumin were associated with decreased fractional and absolute proximal reabsorption, decreased calculated efferent oncotic pressure, and a transitory rise in efferent arteriolar hydrostatic pressure. These findings, however, do not necessarily indicate a predominant role for peritubular "physical factors" in the control of proximal reabsorption. In glomerulonephritic rats a smaller diuresis and an insignificant natriuresis were found after hyperoncotic albumin, especially in anti-GBM nephritis, when no change in SNGFR or proximal absolute reabsorption occurred. In AICN there was a rise in SNGFR and a fall in absolute reabsorptive rate, especially in those nephrons with high filtration rates. There was no evidence that any alteration in efferent arteriolar hydrostatic pressure of calculated efferent oncotic pressure had occurred.