No abstract

Measurements of the water osmotic permeabilities of apical and basolateral membranes of PST cells and of the transepithelial permeability have been carried out using a very fast method with high temporal and spatial resolution. At 25 degrees C the values obtained are: 80.8 +/- 11.9 x 10(-4) cm3/s osmol cm2 of apical (luminal) surface area and 90.1 +/- 13.0 x 10(-4) cm3/s osmol cm2 of basement membrane area (no membrane invaginations taken in account). These values are higher than previously published values due to the use of a faster and more accurate volume measuring and recording system. The transepithelial water osmotic permeability at 25 degrees C is 77 +/- 11 in units of 10(-4) cm3/s osmol cm2 basement membrane area. The transcellular water osmotic permeability is 32 +/- 7 (same units), leaving a paracellular contribution of 45 +/- 10 (same units). In the presence of 2.5 mM parachloromercuribenzenesulfonate (pCMBS) the apical permeability is reduced with an incubation of 10-15 min to 23% of its control value and the basolateral permeability to 8% of its control value (after 25 min) but the transepithelial permeability is only reduced to about 1/2 of the control value. This leaves a transcellular permeability of 6 x 10(-4) cm3/s osmol cm2 of basement membrane area and a paracellular contribution of 33 +/- 6 (same units). These results indicate a significant contribution of the paracellular pathway to the transepithelial water osmotic permeabilities in PST.

Proximal straight tubules were dissected and mounted in a chamber with their lumina occluded. The well-stirred bath could be 95% changed within 84 ms to set up osmotic gradients (delta Coi) across the peritubular cell aspect. Volume changes (less than or equal to 10 pl/mm) were estimated from continuous records of diameter changes (error less than 0.1 micrometers). delta Coi greater than or equal to 2-3 mosM could be discerned. delta Coi values from 10 to 44 mosM were used to evaluate Posc, the cell osmotic water permeability coefficient, and extrapolated to delta Coi = 0. Posc = 25.1 (+/- 2.3) X 10(-4) cm3.s-1.osM-1.cm2 tubular surface area-1. These values are lower than those reported for Pose, the transepithelial osmotic water permeability coefficient, and become lower if corrected for the real (infolded) peritubular cell surface area. Thus, for a given osmotic difference, transcellular water flow finds a higher resistance than paracellular water flow. Experiments were also performed with delta Coi greater than 100 mosM, but interpretation of these data is difficult because of the presence of volume regulatory phenomena and other undesirable effects.

Continuous pathways must pierce the cell membrane to be used by water during osmotic equilibration between proximal straight tubular cells and the external medium, because a) the water osmotic permeability coefficient of the basolateral plasma membrane, Poscb, is high; b) its activation energy, Ea, is as that of free water movement and c) pCMBS inhibits markedly (but reversibly) Poscb and increases Ea to values similar to those observed in lipid bilayers without pores. d) Preliminary measurements of Pd the water diffusive permeability coefficient using NMR indicate that Poscb/Pd is near 4 - 5. The following two observations indicate that a significant paracellular water flow must exist in leaky epithelia. Namely, a) large extracellular solutes are dragged by water in four leaky epithelia: gall bladder, Necturus proximal tubule, rat proximal tubule and Rhodnius malpighian tubule. b) The transcellular water osmotic permeability coefficient is smaller than the transepithelial values available in the rabbit proximal straight tubule. This requires a significant paracellular permeability.

The diffusive water permeability (Pd) of the plasma membrane of proximal kidney tubule cells was measured using a 1H-NMR technique. The values obtained for the exchange time (Tex) across the membrane were independent of the cytocrit and of the Mn2+ concentration (in the range 2.5 to 5 mM). At 25 degrees C the calculated Pd value was (per cm2 of outer surface area without taking into account membrane invaginations) 197 +/- 17 microns/sec. This value equals 22.3 +/- 1.9 microns/sec when the invaginations are taken into account. Cell exposure to 2.5 mM parachloromercuribenzenesulfonic acid, pCMBS, (for 20 to 35 min) reduced Pd to 45% of its control value. Five mM dithiothreitol, DTT, reverted this effect. The activation energy for the diffusive water flux was 5.2 +/- 1.0 kcal/mol under control conditions. It increased to 9.1 +/- 2.2 kcal/mol in the presence of 2.5 mM pCMBS. Using our previous values for the osmotic water permeability (Pos) in proximal straight tubular cells the Pos/Pd ratio equals 18 +/- 1, under control conditions, and 3.2 +/- 0.3 in the presence of pCMBS. These experimental results indicate the presence of pathways for water, formed by proteins, crossing these membranes, which are closed by pCMBS. Assuming laminar flow (within the pore), from Pos/Pd of 13 to 18 an unreasonably large pore radius of 12 to 15 A is calculated which would not hinder cell entry of known extracellular markers. Alternatively, for a single-file pore, 11 to 20 would be the number of water molecules which would be in tandem inside the pore.(ABSTRACT TRUNCATED AT 250 WORDS)

A new method to measure time dependent (t) volume (V) changes in proximal straight tubules (PST) is described. V is calculated from diameter (d) measurements for which a video camera and an integrating circuit are used. A tubular image of high optical contrast is recorded with the TV camera such that the scan lines run crosswise to the tubule. The video signal is analyzed by a special processor which adds 225 tubular diameters of each TV frame and feeds this analog signal to a pen recorder. The fractional error in d measurements is 10(-3). Diameter changes of less than 0.05 micron can be detected, as compared to the usual error of a single measurement of about 0.4 micron. Pcbos, the osmotic water permeability of the contraluminal cell membrane was measured by setting up osmotic steps across it in less than 0.1 s and following the ensuing delta d/delta t. The time delay between solution change and the linear part of the osmotic response was 0.51 +/- 0.05 s. Pcbos was found to be 50.4 (+/- 8.7) X 10(-4) cm3.cm-2 of basement membrane area .s-1.osmolar-1.

Cell osmotic water permeability, Pcos, of the peritubular aspect of the proximal convoluted tubule (PCT) was measured from the time course of cell volume changes subsequent to the sudden imposition of an osmotic gradient, delta Cio, across the cell membrane of PCT that had been dissected and mounted in a chamber. The possibilities of artifact were minimized. The bath was vigorously stirred, the solutions could be 95% changed within 0.1 s, and small osmotic gradients (10-20 mosM) were used. Thus, the osmotically induced water flow was a linear function of delta Cio and the effect of the 70-microns-thick unstirred layers was negligible. In addition, data were extrapolated to delta Cio = 0. Pcos for PCT was 41.6 (+/- 3.5) X 10(-4) cm3 X s-1 X osM-1 per cm2 of peritubular basal area. The standing gradient osmotic theory for transcellular osmosis is incompatible with this value. Published values for Pcos of PST are 25.1 X 10(-4), and for the transepithelial permeability Peos values are 64 X 10(-4) for PCT and 94 X 10(-4) for PST, in the same units. These results indicate that there is room for paracellular water flow in both nephron segments and that the magnitude of the transcellular and paracellular water flows may vary from one segment of the proximal tubule to another.

Osmotic steps, delta C, were produced across the apical cell membrane of isolated rabbit PST by perfusing their lumens with double barreled micropipettes at a rate of 0.5-0.8 nl/s. delta C = 15-46 mOsmolar were induced with mannitol. Changes in luminal diameter were recorded as a function of time with a TV camera and an integrator-processor system with space and time resolutions of 0.03 micron and 0.0167 s (3). The tubules were bathed with oil. Outer tubule diameter was time invariant. Pcaos, the apical cell osmotic permeability was therefore calculated from cell volume changes with time in units of 10(-4) cm3/cm2 X s. Osmolar. Pcaos was independent of delta C. The mean is 22.8 +/- 1.3 (n = 55). With a basolateral permeability of 50.4 (3,12), the transcellular permeability is 14 (same units) smaller than the transepithelial values available. This leads to the conclusion that a significant paracellular water osmotic permeability must exist.