Regulatory volume decrease in a renal distal tubular cell line (A6) II. Effect of Na+ transport rate

Abstract

A6 epithelia, a cell line originating from the distal tubular part of the kidney of Xenopus laevis, were cultured on permeable supports and mounted in an Ussing-type chamber. Cell thickness (Tc), short-circuit current (Isc) and transepithelial conductance (Gt) were recorded while tissues were bilaterally incubated in NaCl solutions and the transepithelial potential was clamped to zero. Effects of inhibition and stimulation of transepithelial Na+ transport on cell volume and on its regulation during a hyposmotic challenge were investigated. Under control conditions a slow spontaneous decrease of Tc described by a linear baseline was recorded. The reduction of the apical osmolality from 260 to 140 mosmol/kg did not alter cell volume significantly, demonstrating a negligible water permeability of the apical barrier. The inhibition of Na+ uptake by replacing apical Na+ by N-methyl-d-glucamine (NMDG+) did not affect cell volume under isotonic conditions. An increase of Tc by 12.1% above the control baseline was recorded after blocking active transport with ouabain for 60 min. The activation of Na+ transport with insulin or oxytocin, which is known to activate the apical water permeability in other epithelia, did not alter cell volume significantly. The insensitivity of cell volume to alterations in apical Na+uptake or Na+ pump rate confirms the close coupling between apical and basolateral transport processes. The blockage of basolateral K+ channels by 5 mM Ba2+ elicited a significant increase in Tc of 16.3% above control. Quinine, a potent blocker of volume-activated K+ channels, did not change Tc significantly. Basolateral hypotonicity elicited a rapid rise in Tc followed by a regulatory volume decrease (RVD). An RVD was also recorded after blocking apical Na+ uptake as well as after stimulating apical Na+ uptake with oxytocin or insulin. Inhibition of active transport with ouabain as well as blocking K+ efflux at the basolateral side with Ba2+ or quinine abolished the RVD. The inhibition of the RVD by ouabain seems to be caused by a depletion of cellular K+, whereas the effects of Ba2+ and quinine are most likely due to the blockage of the basolateral K+ pathway.

Regulatory volume decrease in a renal distal tubular cell line (A6) I. Role of K+ and Cl−

“…If solute excretion occurs concomitantly with water uptake, %Tc Peak will be reduced and T0 will increase with a slower time course. A similar reduction of the rate of rise was observed in experiments with Ba 2+ described in the following paper [6] in which transepithelial Na § transport was enabled. Basolateral Ba 2 § [23] and an increase in apical Na § uptake [22] depolarize the intracellular potential.…”

Forskolin increases apical sodium conductance in cultured toad kidney cells (A6) by stimulating membrane insertion

“…The lack of cell volume increase upon the drastic reduction of π ap shows that the apical membrane is impermeable to water even when cAMP production is stimulated by forskolin. The transient response in cell volume caused by reducing π bl resembles that found in previous studies from our laboratory [7]. It demonstrates that the basolateral membrane is highly permeable to water and that the expansion of cell volume triggers a regulatory volume decrease in A6 cells.…”

“…In this model the probability of a vesicle being in the apical membrane is (Eq. 7): P mem =k ins /(k ret +k ins ) (7) Assuming that in the steady state k ins =k ret and that agonists modify apical membrane area M a by acting only on either one or both rate constants k ins and k ret we can make specific predictions on the additivity of the effects of agonists. According to the model, if two agonists promote membrane insertion from the same intracellular pool, maximal doses of two agonists that increase k ins will not have additive effects on C T .…”

A pathophysiologic study of the kidney tubule to optimize organ preservation solutions

“…In the context of ischemic depletion of ATP, energy dependent solute transporters such as the Na + /K + ATPase are no longer able to regulate cell volume. An accumulation of intracellular Na + is followed by influx of anions to maintain electroneutrality [25][26][27] and water follows osmotically into the cell. The use of strophanthidin in our system to block the Na + /K + pump modeled the physiologic response of tubules to starvation of ATP with regard to cell volume control.…”