E. González

Effect of para-chloromercuribenzenesulfonic acid and temperature on cell water osmotic permeability of proximal straight tubules.

The apparent Arrhenius energy of activation (Ea) of the water osmotic permeability (Pcos) of the basolateral plasma cell membrane of isolated rabbit proximal straight tubules has been measured under control conditions and after addition of 2.5 mM of the sulfhydryl reagent, para-chloromercuribenzenesulfonic acid (pCMBS), of mersalyl and of dithiothreitol. Ea (kcal/mol) was 3.2 +/- 1.4 (controls) and 9.2 +/- 2.2 (pCMBS), while Pcos decreased with pCMBS to 0.26 +/- 0.17 of its control value. Mersalyl also decreased Pcos both in vitro and in vivo (using therapeutical doses). These actions of pCMBS and mersalyl were quickly reverted with 5 mM dithiothreitol and prevented by 0.1 M thiourea. Ea for free viscous flow is 4.2 and greater than 10 for non-pore-containing lipid membranes. By analogy with these membranes and with red blood cells, where similar effects of pCMBS on Pos are observed, it is concluded that cell membranes of the proximal tubule are pierced by aqueous pores which are reversibly shut by pCMBS. Part of the action of mercurial diuretics can be explained by their action on Pcos.

Pathways for volume flow and volume regulation in leaky epithelia.

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 increasesEa to values similar to those observed in lipid bilayers without pores. d) Preliminary measurements ofPd the water diffusive permeability coefficient using NMR indicate thatPoscb/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 andRhodnius 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.

Effect of transtubular osmotic gradients on the paracellular pathway in toad kidney proximal tubule

SummaryWe have evaluated with the aid of electron microscopy changes in the width of the paracellular pathway and in the magnitude of the paracellular passage of lanthanum ions when a transtubular osmotic gradient was established by making the tubular lumen hyperosmotic with 50 mM urea or mannitol. Both transtubular gradients (with urea or mannitol) induce changes in the ultrastructure of the tight junction. The changes are characterized by widening of the tight junction and the “disappearance” of the substance forming the intermediate line. Urea has a larger effect than mannitol. The magnitude of lanthanum crossing extracellularly through the tubular epithelium also increases under the urea induced transtubular gradient.

The proximal straight tubule (PST) basolateral cell membrane water channel: Selectivity characteristics

Proximal straight tubules (PST) were dissected from rabbit kidneys, held by crimping pipettes in a chamber and bathed in a buffered isosmotic (295 mOsm/kg) solution containing 200 mm mannitol (MBS). Changes in tubule diameter were monitored on line with an inverted microscope, TV camera and image processor. The PST were then challenged for 20 sec with MBS made 35 mOsm/kg hyperosmotic by addition of either NaCl, KCl, mannitol (M), glycerol (G), ethylene glycol (E), glycine (g), urea (U), acetamide (A) or formamide (F). With NaCl, KCl, M, G, E, g, U, and A, tubules shrunk osmometrically within 0.5 sec and remained shrunk for as long as 20 sec without recovering their original volume (sometimes A showed some recovery). PST barely shrunk with F and quickly recovered their original volume. The permeability coefficients were 0 μm/sec (NaCl, M, g, E and U), 1 μm/sec (A), 84 μm/sec (F) and 0.02 μm/sec (G). The reflection coefficients σ = 1.0 (NaCl, KCl, M, G, E, g and U), 0.95 (A) and 0.62 (F). Similar σ values were obtained by substituting 200 mOsm/kg M in MBS by either NaCl, KCl, G, E, g, U, a or F. The olive oil/water partition coefficients are 5 (M), 15 (U), 85 (A) and 75 (F) (all x10−5). Thus, part of F permeates the cell membrane through the lipid bilayer. The probing molecules van der Waals diameters are 7.4×8.2×12.0 (M), 3.6×5.2×5.4 (U), 3.8×5.2 ×5.4 (A) and (3.4×4.5×5.4 (F) Å. We conclude that only F clearly permeates the water channel (WCH). Water molecules must single file within the WCH. After subtraction of the bilayer permeability of the probes, we estimate for the WCH selectivity filter cross-section a diameter of 4.2–4.7 Å (if it is circular) and 3.6×4.2 Å (if it is rectangular). But if the oxygens facing the WCH lumen H bond with the molecules crossing the WCH, the WCH selectivity filter would be 3.3–3.8 Å (circular) and 3.6×4.0 Å (rectangular).

Fluid secretion in Rhodnius upper malpighian tubules (UMT): water osmotic permeabilities and morphometric studies.

We have measured the osmotic permeability of the basolateral cell membrane (Poscb) and compared it with the transepithelial permeability (Poste) to calculate the paracellular (Posp) permeability of the upper malpighian tubules (UMT) of the 5th instar of Rhodnius prolixus under several experimental conditions, namely, at rest and after stimulation to secrete with 5-HT, each under control conditions (no treatment), after treatment with pCMBS, and after addition of pCMBS and DTT. Secretion rate is negligible at rest. During stimulation mean secretion rate is 43.5 nl/cm2 sec. Secretion is severely curtailed by pCMBS and fully restored by DTT. Poscb = 9.4 (resting, control); 5.8 (control + pCMBS); 10.7 (control + pCMBS + DTT); 20.6 (stimulated, control); 14.7 (stimulated + pCMBS); 49.1 (stimulated + pCMBS + DTT) (x10?4 cm3/cm2 sec Osm). Calculated Posp are higher than the transcellular permeability, Posc, at rest and after stimulation. Electron micrograph morphometry of UMT sections show that cells significantly decrease their volume after stimulation. Lateral intercellular space (LIS) and basolateral extracellular labyrinth (BEL) are barely discernible at rest. LIS and BEL are widely dilated in stimulated UMT. Thus, ions have restricted access to the deep and narrow basolateral cell membrane indentations at rest, but they have ready access to cell membrane indentations after stimulation, because of the opening of LIS and BEL. These findings are discussed in relation to isosmotic secretion. The rate-limiting step for paracellular movement is located at the smooth septate junctions.

Length of the selectivity filter of aquaporin‐1*

We have characterized the selectivity filter of the water channel aquaporin‐1 (AQP1) of proximal straight tubules (PST), as an equivalent cylindrical structure with a diameter of ∼ 4.5 Å, where water molecules single file. We report here efforts to evaluate its length. PST were dissected from rabbit kidneys, held with pipettes in a chamber bathed in a buffered mannitol isosmotic solution (MBS, 295 mOsm/kg). Changes in tubule cell volume with time (dV/Adt), were monitored, on line, with an inverted microscope, a TV camera and an image processor. Osmotic permeability coefficients, Pos, and reflection coefficients (σs) were measured with several solutes: mannitol (M), raffinose (R), sucrose (S), glycerol (G), acetamide (A) and urea (U). For this purpose PST were suddenly exposed (in ∼ 80 ms and for 20 s) to a hyperosmolality step (ΔCs) achieved by adding to MBS a ΔCs of 35 mOsm/kg of either R, S, M, G, A or U. Cells shrunk within 500 ms of t = 0 to their osmometric volume and remained shrunk for the 20 s of the ΔCs. Pos was measured from the shrinking curves; Pos = 3000 ± 25 μm/s with either R, S, M, G, A or U. This procedure also allowed to calculate σs; σs = 1.00 for R, S, M, G, A and U, indicating that these solutes do not penetrate the water channel. In contrast, the shrinking curves produced by a ΔCs = 35 mOsm/kg formamide (F) were 15th to 16th slower and smaller (subosmometric) than those produced by a ΔCs = 35 mOsm/kg of R, S, M, G, A or U. Furthermore, with F, cells did not remain shrunk. They recovered their original volume within 3 s. Pos (measured with F) is denoted as Pos*; Pos* = 480 ± 30 μm/s. σs,formamide (denoted σsp) = 0.16 ± 0.01. Use of σsp and Pos* values in Hills equations for the bimodal theory of osmosis leads to n = 2–3, n being the number of water molecules single filing within the channel selectivity filter, whose length must lie within 6 to 9 Å, a value lower than previous values calculated from the Pos Pd* ratio.

Effect of hypertonic urea and mannitol on distal nephron permeability

SummaryThe paracellular pathway permeability is known to increase in perfused amphibian kidneys if the luminal fluid is made hyperosmotic with mannitol or urea. To investigate whether luminal hypertonicity increases paracellular pathway permeability in the mammalian nephron, early rat distal tubules were micropunctured and perfused through one micropipette with either isosmotic saline (IS), hyperosmotic urea (HU) or hyperosmotic mannitol (HM) solutions. A second micropipette was placed down-stream in the same tubule and test solutions of 30 nl of a mixture of14C-inulin and3H-mannitol or of3H-inulin and14C-urea were injected. Similar intratubular injections of tracers were performed in a second group of rats undergoing diuresis induced either by infusing intravenously saline alone (VS) or receiving saline plus 0.4 M urea (VU). In the latter group (VU) luminal urea concentration was increased without the tubular lumen being made hyperosmotic to its peritubular fluid.Urinary inulin recovery was essentially complete and unaffected by experimental procedures. Difference between mannitol recoveries in isosmotic saline and hyperosmotic urea perfusions IS-HU was 2.6±0.8% (P<0.001). Difference in urea recoveries IS-HM was 4.1±5.1% (P>0.40), IS-HU was 13.9±5.3% (P=0.015) and, VS-VU=17.0±3.4 (P<0.001). Therefore, elevated luminal urea concentration increased tracer mannitol and also tracer urea permeability, both in the presence and absence of tubular hyperosmolarity. Electron microscopic observations showed changes in geometry of tubular junctional complexes compatible with the observed increase in permeability.

Abnormal transtubular permeability to raffinose during intravenous infusion of urea and of mannitol in the intact dog kidney

Serial urine samples were collected every 3 s after a bolus injection containing labelled raffinose plus a “glomerular substance” (i.e. a substance eliminated only by filtration like inulin or dextran MW 15,000–17,000) into the renal artery of dogs. Three groups of dogs were explored. They underwent profuse diuresis subsequent to i.v. infusion of saline alone (S), saline + urea (U) or saline + mannitol (M) respectively. The amount of raffinose and of “glomerular substances” eliminated in the urine was plotted as a function of a normalizedurine volume. Aurine volume of 100% is defined as the urinary volume collected from the time of injection up to the moment in which urinary concentration of the glomerular substance reaches 50% of its peak concentration. In the three groups, elimination of “glomerular substances” began 50–60 s after the bolus injection corresponding to aurine volume of 65.9±0.9%. In the S group raffinose was eliminated after aurine volume of 54.7%. A fraction equal to (3.5±1.0)×10−4 of the amount of raffinose injected in the bolus preceeded elimination of “glomerular substances”. In the U group, a fraction equal to (21.9±5.3)×10−4 of the amount of raffinose injected preceeded climination of “glomerular substances”. This value is statistically higher than that obtained in the S group. In the U group, raffinose was eliminated 15–20 s after injection, corresponding to aurine volume of 18.8%. This urine volume is not different from that occupied by catheter, ureter and renal pelvis. It is concluded that raffinose, is eliminated ahead of dextran in the U group because a small but significant amount of raffinose crosses the tubular wall. Thus, urea infusion opens the paracellular pathways since raffinose stays in the extracellular space. Animals of the M group also showed an early appearance of raffinose although at a less distal level of the nephron.