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This paper describes experiments designed to evaluate Na(+) and Cl(-) transport in isolated proximal straight tubules from rabbit kidneys. When the perfusing solution was Krebs-Ringer buffer with 25 mM HCO(3) (-) (KRB) and the bath contained KRB plus 6% albumin, net volume reabsorption (J(v), nl min(-1) mm(-1) was -0.46 +/- 0.03 (SEM); V(e), the spontaneous transepithelial potential difference, was -1.13 +/- 0.05 mV, lumen negative. Both J(v), and V(e), were reduced to zero at 21 degrees C or with 10(-4) M ouabain, but J(v), was not HCO(3) (-) dependent. Net Na(+) reabsorption, measured as the difference between (22)Na(+) fluxes, lumen to bath and bath to lumen, accounted quantitatively for volume reabsorption, assuming the latter to be an isotonic process, and was in agreement with the difference between lumen to bath (22)Na(+) fluxes during volume reabsorption and at zero volume flow. The observed flux ratio for Na(+) was 1.46, and that predicted for a passive process was 0.99; thus, Na(+) reabsorption was rationalized in terms of an active transport process. The Cl(-) concentration of tubular fluid rose from 113.6 to 132.3 mM during volume reabsorption. Since V(e), rose to +0.82 mV when tubules were perfused with 138.6 mM Cl(-) solutions, V(e) may become positive when tubular fluid Cl(-) concentrations rise during volume reabsorption. The permeability coefficients P(Na) and P(Cl) computed from tracer fluxes were, respectively, 0.23 x 10(-4) and 0.73 x 10(-4) cm s(-1). A P(Na)/P(Cl) ratio of 0.3 described NaCl dilution potentials at zero volume flow. The magnitudes of the potentials were the same for a given NaCl gradient in either direction and P(Na)/P(Cl) was constant in the range 32-139 mM NaCl. We infer that the route of passive ion permeation was through symmetrical extracellular interfaces, presumably tight junctions, characterized by neutral polar sites in which electroneutrality is maintained by mobile counterions.

The present experiments were designed to evaluate the effects of varying the osmolality of luminal solutions on the antidiuretic hormone (ADH)-independent water and solute permeability properties of isolated rabbit cortical collecting tubules. In the absence of ADH, the osmotic water permeability coefficient (cm s(-1)) P(f) (l-->b), computed from volume flows from hypotonic lumen to isotonic bath, was 20 +/- 4 x 10(-4) (SEM); the value of P(f) (b-->l) in the absence of ADH, computed from volume flows from isotonic bath to hypertonic lumen, was 88 +/- 15 x 10(-4) cm s(-1). We also measured apparent urea permeability coefficients (cm s(-1)) from (14)C-urea fluxes from lumen to bath (P(DDurea) (l-->b)) and from bath to lumen (P(DDurea) (b-->l)). For hypotonic luminal solutions and isotonic bathing solutions, P(DDurea) (l-->b) was 0.045 +/- 0.004 x 10(-4) and was unaffected by ADH. The ADH-independent values of P(DDurea) (l-->b) and P(urea) (b-->l) were, respectively, 0.216 +/- 0.022 x 10(-4) cm s(-1) and 0.033 +/- 0.002 x 10(-4) cm s(-1) for isotonic bathing solutions and luminal solutions made hypertonic with urea, i.e., there was an absolute increase in urea permeability and asymmetry of urea fluxes. Significantly, P(DDurea) (l-->b) did not rise when luminal hypertonicity was produced by sucrose; and, bathing fluid hypertonicity did not alter tubular permeability to water or to urea. We interpret these data to indicate that luminal hypertonicity increased the leakiness of tight junctions to water and urea but not sucrose. Since the value of P(f) (b-->l) in the absence of ADH, when tight junctions were open to urea, was approximately half of the value of P(f) (l-->b) in the presence of ADH, when tight junctions were closed to urea, we conclude that tight junctions are negligible paracellular shunts for lumen to bath osmosis with ADH. These findings, together with those in the preceding paper, are discussed in terms of a solubility-diffusion model for water permeation in which ADH increases water solubility in luminal plasma membranes.

We have used rat cortical collecting tubules perfused in vitro to study the effects of antidiuretic hormone (ADH) and desoxycorticosterone (DOCA) on the unidirectional fluxes of sodium. We found that in the basal state, lumen-to-bath flux (Jlb) and bath-to-lumen flux (Jbl) of 22Na were approximately equal, 39.5 +/- 3.9 and 41.8 +/- 11.0 pmol X min-1 X min-1, respectively, resulting in no net flux. Addition of 100 microU/ml ADH to the bath produced a stable increase in Jlb to 58.3 +/- 4.7 pmol X min-1 X mm-1. Pretreatment of the animal with DOCA for 4 to 7 d (20 mg/kg per d) increased baseline Jlb to 81.6 +/- 8.7 pmol X min-1 X mm-1. Addition of ADH to a tubule from a DOCA-pretreated rat caused an increase in Jlb to 144.1 +/- 12.0 pmol X min-1 X mm-1 X Neither hormone had an effect on Jbl X Thus ADH produced a greater absolute and fractional increase in Jlb when the animal was pretreated with DOCA, and the ADH-induced increase over baseline was greater than the DOCA-induced increase. Both the ADH-and DOCA-induced stimulation of Jlb were completely abolished by 10(-5) M luminal amiloride, suggesting that the route of sodium transport stimulated by both hormones involves apical sodium channels. However, ADH and DOCA have very different time courses of action; ADH acted within minutes, while aldosterone and DOCA are known to require 90-180 min. The facilitating action of ADH on DOCA-induced stimulation of sodium transport may be important for maximal sodium reabsorption and for the ability to achieve a maximally concentrated urine.

The present studies tested the hypothesis that osmotic disequilibrium between luminal and peritubular fluids is the driving force for net volume absorption in the isolated proximal straight tubule. Isolated tubule segments from superficial rabbit renal cortex were perfused at varying rates with a high chloride and bicarbonate-free solution as they were bathed with a normal bicarbonate-Krebs-Ringer buffer solution at 38 degrees C. Increasing the perfusion rate from congruent to 4 to congruent to 30 nl/min produced a monotonic increase in net volume absorption (Jv) from 0.18 +/- (sem) 0.03 to 0.62 +/- 0.08 nl . min-1. The chloride concentration in collected fluid samples rose from congruent to 137 to congruent to 147 mEq/liter over the same perfusion rate range. Ouabain (10(-4) m) added to the bathing solution inhibited Jv by a rate which varied from 0.20 to 0.28 nl . min-1 . min-1, depending on the perfusion rate. A mathematical model of the axial flows and transepithelial transport processes was developed. This model, and the experimental data, is consistent with the view that the driving force for isotonic fluid absorption in these tubules depends on the axial maintenance of osmotic disequilibrium between the perfusate and the bathing solution. Increasing the perfusion rate opposes osmotic equilibration by minimizing the extent to which dissipative fluxes of chloride and bicarbonate ions change the transepithelial chloride and bicarbonate concentration gradients, and by minimizing the tendency of the luminal cryoscopic osmolality to increase as volume absorption occurs.

The effect of arginine vasopressin (ADH) on water permeability and transepithelial voltage was examined in cortical collecting tubules from a specific pathogen-free line of male Sprague-Dawley rats (75-125 g body weight). Tubules were bathed in a medium resembling serum ultrafiltrate (310 mOsm/kg H2O) at 38 degrees C. Osmotic water permeability (Pf, micron/sec) was determined by the volume flow occurring with a hypo-osmotic perfusate (210-220 mOsm/kg H2O) and diffusional water permeability (Pd, micron/sec) was calculated from the lumen-to-bath flux of tritiated water using an isosmotic perfusate. In the absence of ADH, both Pf and Pd were low, 17 +/- 6 and 9.0 +/- 0.6 (SEM), respectively. ADH added to the bath at concentrations above 0.5 microunits/ml increased Pf, with a maximal response at 40 microunits/ml or greater. With 100 microunits/ml ADH, Pf and Pd were, respectively, 994 +/- 117 and 37.0 +/- 2.4. Without ADH, the transepithelial voltage was variable (range, -5.4 to +2.5 mV; mean, -1.9 +/- 0.4); however, with 100 microU/ml ADH, it hyperpolarized (lumen-negative) by 4.2 +/- 0.8 mV. In contrast to findings in the rabbit, both the hyperpolarization and the increased water permeability persisted for at least 3 hr. The higher water permeabilities are consistent with the shorter length of the cortical collecting tubule in the rat, and may reflect the importance of attaining osmotic equilibration within the cortex during maximal antidiuresis.

The present experiments were designed to evaluate the effective thickness of the unstirred layers in series with native and porous (i.e., in the presence of amphotericin B) lipid bilayer membranes and, concomitantly, the respective contributions of membranes and unstirred layers to the observed resistances to the diffusion of water and nonelectrolytes between aqueous phases. The method depended on measuring the tracer permeability coefficients for the diffusion of water and nonelectrolytes (P(DDi), cm sec(-1)) when the aqueous phase viscosity (eta) was increased with solutes having a unity reflection coefficient, such as sucrose or dextran. The effective thickness of the unstirred layers (alpha(t), cm) and the true, or membrane, permeability coefficients for diffusion of water and nonelectrolytes (P(mmi), cm sec(-1)) were computed from, respectively, the slope and intercept of the linear regression of 1/P(DDi) on eta. In both the native and porous membranes, alpha(t) was approximately 110 x 10(-4) cm. The ratio of P(f), the osmotic water permeability coefficient (cm sec(-1)) to P(mmH2O) was 1.22 in the native membranes and 3.75 in the porous membranes. For the latter, the effective pore radius, computed from Poiseuille's law, was approximately 5.6 A. A comparison of P(mmi) and P(DDi), indicated that the porous membranes accounted for 16, 25, and 66% of the total resistance to the diffusion of, respectively, H(2)O, urea, and glycerol, while the remainder was referable to the unstirred layers.

The present experiments were designed to evaluate coupling of water and nonelectrolyte flows in porous lipid bilayer membranes (i.e., in the presence of amphotericin B) in series with unstirred layers. Alterations in solute flux during osmosis, with respect to the flux in the absence of net water flow, could be related to two factors: first, changes in the diffusional component of solute flux referable to variations in solute concentrations at the membrane interfaces produced by osmotic flow through the unstirred layers; and second, coupling of solute and solvent flows within the membrane phase. Osmotic water flow in the same direction as solute flow increased substantially the net fluxes of glycerol and erythritol through the membranes, while osmotic flow in the opposite direction to glycerol flow reduced the net flux of that solute. The observed effects of osmotic water flow on the fluxes of these solutes were in reasonable agreement with predictions based on a model for coupling of solute and solvent flows within the membrane phase, and considerably in excess of the prediction for a diffusion process alone.