K+ transport in the mesonephric collecting duct system of the toad Bufo bufo : microelectrode recordings from isolated and perfused tubules
August Krogh Institute, Department of Zoophysiology, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark
* Author for correspondence (e-mail: nmobjerg{at}aki.ku.dk )
Accepted 7 January 2001
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Summary |
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Key words: amphibian, Ba2+, Bufo bufo, collecting duct, collecting tubule, K+ conductance, K+ secretion, kidney, mesonephros, ouabain, toad
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Introduction |
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The structural and functional unit of the kidney, the nephron, can in the
amphibian mesonephros be divided into seven morphological and functional
distinct sections: Malpighian corpuscle, neck segment, proximal tubule,
intermediate segment, early distal tubule, late distal tubule and, finally,
the collecting tubule, which opens into collecting ducts that lead the urine
to the ureter (Dantzler, 1992;
Dietl and Stanton, 1993
;
Hentschel and Elger, 1989
;
Møbjerg et al., 1998
).
The heterocellular epithelium constituting the collecting tubules and
collecting ducts here referred to as the collecting duct system
takes part in the final adjustment of the urine.
Most studies on amphibian renal tubules have been carried out on aquatic
urodeles, because they have a large tubule diameter and large cell size
(Richards and Walker, 1937;
Stoner, 1977
). In particular,
the kidney of Amphiuma has been used as a model for vertebrate distal
tubule transport studies (for a review, see
Dietl and Stanton, 1993
). Based
on data obtained mainly from urodeles, the collecting tubules have been shown
to display a lumen-negative voltage, to reabsorb Na+ and to secrete
K+ (Horisberger et al.,
1987
; Hunter et al.,
1987
; Stoner,
1977
; Wiederholt et al.,
1971
). However, little is known about the function of the
collecting tubule and especially the collecting ducts of anuran amphibians.
The aim of our study was to characterize K+ transport by the cells
within these tubule segments of a terrestrial anuran, the common European toad
Bufo bufo. For this purpose we isolated and perfused tubules in
vitro and impaled cells with conventional microelectrodes.
Since the early work by Richards and Walker
(1937), who introduced the
micropuncture technique in renal tubules from kidneys of amphibians, several
investigators have demonstrated the ability of the distal nephron from urodele
amphibians to secrete K+ (Bott,
1962
; Garland et al.,
1975
; Wiederholt et al.,
1971
). These data were subsequently confirmed in a study on
isolated and perfused tubules from the distal nephron of both urodele and
anuran amphibians (Stoner,
1977
). In an electrophysiological study on in vitro
perfused collecting tubules from Amphiuma sp., it was subsequently
shown that the heterocellular epithelium of the collecting tubule secretes
K+ (Horisberger et al.,
1987
). These workers, however, could not find a significant
K+ conductance in the luminal cell membrane of the collecting
tubule of Amphiuma, and therefore argued that K+ secretion
could not occur across the luminal membrane
(Horisberger et al., 1987
;
Hunter et al., 1987
). Thus, in
the light of these observations it has been postulated that K+
secretion by the amphibian collecting tubule occurs via a passive,
paracellular mechanism (Dietl and Stanton,
1993
). Therefore, it was believed that K+ secretion in
amphibians differs from secretion by the cortical collecting tubule in mammals
(for reviews, see Giebisch,
1998
; Palmer,
1999
).
Nevertheless, Stoner and Viggiano
(1998) recently used patch
clamp and characterised maxi K+ channels from cell-attached patches
on the apical membrane of the everted collecting tubule of aquatic-phase tiger
salamanders Ambystoma tigrinum. K+ adaptation in the
salamanders caused a tenfold increase in the number of detectable maxi
K+ channels. After K+ adaptation the maxi K+
channels contributed to the ability of the collecting tubule in A.
tigrinum to secrete K+
(Stoner and Viggiano, 1999
).
Stoner and Viggiano (2000
)
have also provided evidence for an apical intermediate conductance,
amiloride-insensitive, non-specific cation channel in the collecting tubule of
the tiger salamander. Since the channel is observed most frequently in
K+-adapted animals it may be important in K+
secretion.
These contradictory findings on the mechanism by which K+ is secreted by the collecting tubule led us to explore the means of K+ transport by the collecting duct system of the mesonephric kidney of Bufo bufo, an anuran species that in its adult life is fully adapted to the terrestrial environment.
In the present study on isolated and perfused collecting tubules and collecting ducts, microelectrode recordings demonstrate the presence of a large K+ conductance in the basolateral cell membrane. Furthermore an apical K+ conductance is present in both collecting tubules and collecting ducts. Thus, this study provides evidence that is in agreement with a transcellular secretion of K+ in the collecting duct system of the toad.
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Materials and Methods |
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Collecting tubules and collecting ducts were dissected from the mesonephros
of adult toads. The morphology and ultrastructure of the mesonephric kidney of
Bufo bufo has been described in a previous study
(Møbjerg et al., 1998).
Only female toads were used for the present study, as they lack the
communication between the mesonephric kidney and gonadal system, which in
males is used for sperm transport.
The animals were killed by decapitation and the kidneys were immediately excised and cut into transverse sections approximately 1 mm thick using razor blades. The dissection medium contained (in mmol l-1): 96.8 Na+, 3.0 K+, 1.8 Ca2+, 1.0 Mg2+, 81.6 Cl-, 20.0 HCO3-, 1.0 SO42-, 0.8 HPO42-, 0.2 H2PO4-, 5.5 glucose, 3.3 glycine, 0.4 PVP (polyvinyl-pyrolidone), 5.0 Hepes, titrated to pH 7.8 with NaOH.
Tubules were identified under a stereomicroscope during free-hand dissection with sharpened forceps and needles (Terumo, Neolus 0.4x20 mm) at 6°C. Collecting ducts were isolated from the dorsal zone of the kidney beneath the connective tissue capsule. Collecting tubules run ventrally from the transition zone with the ducts. The collecting ducts had an outer tubule diameter of approximately 60-70 µm (Fig. 1A,B). The outer diameter of the collecting tubules was approximately 50-60 µm (Fig. 1C,D). The dissected length of the tubules was in the range 300-500 µm.
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The isolated tubules were transferred in a small volume of dissection medium to a bath chamber mounted on an inverted microscope equipped with Nomarski optics (Zeiss, Axiovert 135 TV) and perfused in vitro at room temperature. The control solution, which was used to perfuse the bath and the lumen of the tubules, consisted of (in mmol l-1): 101.8 Na+, 3.0 K+, 1.8 Ca2+, 1.0 Mg2+, 81.6 Cl-, 25.0 HCO3-, 1.0 SO42-, 0.8 HPO42-, 0.2 H2PO4-, equilibrated with 1.8 % CO2 in O2, pH 7.8. In high [K+] solutions, the K+ concentration was raised to 20 mmol l-1 by equimolar substitution with Na+. In experiments with Ba2+, 1 mmol l-1 BaCl2 was added to the control solution, or the high [K+] solution. Ouabain and furosemide were used in control solution at a final concentration of 1 mmol l-1 and 10-5 mmol l-1, respectively.
In order to hold the concentric pipettes used for tubule perfusion we used
the system (Luigs & Neumann, Germany) described by Greger and Hampel
(1981) for in vitro
perfusion of isolated renal tubules, which was modified from the original
system described by Burg et al.
(1966
). The pipette arrangement
consisted, on the perfusion side, of a constricted holding pipette and a
single-barrelled perfusion pipette inserted into the tubule lumen
(Fig. 1A,C). A small glass
capillary in the perfusion pipette insured fast fluid exchange (o.d. 0.3 mm,
i.d. 0.2 mm, Drummond Scientific Company, PA, USA). The tubule was held by a
holding pipette on the fluid-collection side. The dimensions of the pipettes
were made to fit the tubules using as SM II/1 Puller from Luigs & Neumann
(Germany). Holding pipettes (o.d. 2.1 mm, i.d. 1.6 mm) and perfusion pipettes
(o.d. 1.2 mm, i.d. 1.0 mm) were made from glass tubing from Drummond
Scientific Company (PA, USA).
Cells were impaled with microelectrodes mounted on a Leitz micromanipulator
(Germany) and the basolateral cell membrane potential
(Vbl) was recorded with respect to the grounded bath
(Fig. 1D). Impalements were
achieved by placing the microelectrode tip against the basal surface of the
cell and gently tapping the manipulator or the table. The electrodes were
pulled from borosilicate glass with filament (Clark Electromedical, UK) on a
vertical electrode puller (Narishige, Japan). When filled with 1 mol
l-1 KCl they had a resistance of 100-150 M. The recording of
Vbl was accepted if the impalement was achieved by a
sudden change in the potential read by the electrode and if it was stable and
lasted longer than 30 s. For the intracellular voltage recordings we used a
WPI Duo 773 electrometer (World Precision Instruments, FL, USA) interfaced
with a PowerLab/4s recording unit using a 16-bit analog-to-digital converter
for digitization (ADInstruments, NSW, Australia). The software, Chart, which
comes with the PowerLab has a 12-bit resolution. The sampling rate was 40
s-1.
Figures were made in Origin 6.0 (Microcal, MA, USA) and imported to CorelDRAW 9 (Corel Corporation, Canada) for the graphic presentation. The results are presented as original recordings made by the PowerLab/4s recording unit together with the Chart v3.4.11 application program. Values are given as mean ± S.E.M. (standard error of the mean), where N is equal to the number of cells impaled in each experiment. Statistics were performed using two-tailed paired or non-paired t-tests as appropriate and a significance level of 0.05 was applied.
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Results |
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Cell membrane voltage
For the following experiments we dissected collecting tubules and
collecting ducts, which both consist of a heterocellular epithelium comprising
principal cells and intercalated cells.
Fig. 2 is a frequency
distribution of Vbl in 120 cells from collecting ducts
(N=50) and collecting tubules (N=70). Cells from the
collecting ducts seemed to fall into two groups; one group was relatively more
hyperpolarized, with a Vbl averaging -80 to -85 mV and the
other being more depolarized, Vbl averaging approximately
-65 mV (Fig. 2A). The
Vbl of cells from collecting tubules often fell inbetween
the two collecting duct groups, averaging -70 to -75 mV
(Fig. 2B). The results
presented below are based on 55 impalements made on 37 tubules from 31 toads.
It was possible to distinguish between intercalated cells and principal cells
at magnifications from 200x using Nomarski optics
(Fig. 1B). The apical surface
of the intercalated cells possesses microvilli and bulges slightly into the
lumen of the tubule, whereas the apical surface of the principal cells appears
smooth. However, it was impossible to visualize the tip of the electrode and
the impaled cell could therefore not be determined with absolute certainty. So
far we have found no functional tests that could distinguish principal cells
and intercalated cells in collecting tubules or collecting ducts, as for
example reported by Schlatter and Schafer
(1987) for the cortical
collecting tubule of the rat. All cells behaved similarly with respect to
K+ conductance (see below).
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Effect of K+ concentration steps
We examined the presence of basolateral and luminal K+
conductances in cells from collecting tubules and collecting ducts. Bath and
luminal [K+] were changed from 3 mmol l-1 to 20 mmol
l-1. The [K+] changes in both bath and luminal solutions
resulted in a depolarization of Vbl
(Fig. 3A,D).
|
As illustrated in Fig. 3B,C the basal [K+] step depolarized Vbl in cells from both collecting ducts and collecting tubules. In 15 cells from 12 collecting ducts with a mean Vbl of -74±4 mV depolarization was 36±3 mV, and in 23 cells from 15 collecting tubules with a mean Vbl of -66±2 mV the depolarization was 30±2 mV.
Notably, Vbl depolarized in cells from both collecting tubules and collecting ducts in response to luminal [K+] steps. Fig. 3E,F summarizes the luminal K+ substitution experiments from 22 cells. In 11 cells from 9 collecting ducts with a mean Vbl of -73±3 mV depolarization was 16±3 mV, and in 11 cells from 7 collecting tubules with a mean Vbl of -70±3 mV, Vbl depolarized by 11±3 mV. The luminal and basal K+ substitution experiments indicate that there is a luminal and a basolateral K+ conductance in cells of both collecting tubules and collecting ducts.
Effect of Ba2+
In the next series of experiments we tested the effect of Ba2+,
an inhibitor of several types of K+ channels
(Greger and Gögelein,
1987). In Fig. 4A a
voltage trace from a collecting tubule cell illustrates the effect of 1 mmol
l-1 BaCl2 in bath control solution and in 20 mmol
l-1 [K+] on Vbl. A summary of
experiments from eight cells originating from four collecting ducts and three
collecting tubules with a mean Vbl of -64±4 mV is
presented in Fig. 4B. Upon
addition of Ba2+ to the bath control solution,
Vbl depolarized by 30±6 mV. The depolarizing effect
on Vbl by the high [K+] test solution was
inhibited by Ba2+.
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In Fig. 5A a voltage trace from a collecting tubule cell shows the effect of perfusing the tubule lumen with Ba2+ on Vbl. In this cell, Ba2+ in the control solution had no effect on Vbl, although luminal high [K+] solution elicited a depolarization of Vbl. However, the inhibitor abolished the depolarization by the high [K+] solution. One likely explanation is that, in the most hyperpolarized cells, the K+ conductance of the basolateral cell membrane dominates the total cell conductance. Vbl was therefore not affected by the inhibition of the apical K+ conductance with Ba2+. The apical K+ conductance was nevertheless revealed in the presence of a basolateral [K+] step. Fig. 5B shows the effect of luminal perfusion with Ba2+ in the same cell in an experiment where the cell was depolarized by a basally applied high [K+] solution. The high [K+] solution depolarized Vbl by 32 mV and a further depolarization of 8 mV was observed after the luminal perfusion with Ba2+.
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A summary of experiments from four collecting tubule cells and one collecting duct cell originating from five tubules showing the effect of luminal Ba2+ is presented in Fig. 5C. In these five cells Ba2+ depolarized a Vbl of -71±5 mV by 9±3 mV. Fig. 5D is a summary of three experiments made on two collecting tubule cells and one collecting duct cell, showing the effect on Vbl of luminal perfusion with Ba2+ in control solution, while depolarizing the cell with a basal high [K+] solution. Ba2+ had an effect if high [K+] in the bath left the cell relatively hyperpolarized.
Taken together, experiments with the K+-channel inhibitor Ba2+ provide further evidence for the presence of K+ channels in the both luminal and basolateral cell membranes.
Effect of basolateral inhibitors
In order to test the mechanism by which K+ enters the cell
across the basolateral cell membrane, transport inhibitors were added to the
bath solution. A voltage trace illustrating the effect on
Vbl of 1 mmol l-1 ouabain in the bath control
solution is shown in Fig. 6A.
Addition of ouabain to the bath solution for 2-4 min resulted in a rapid and
fully reversible depolarization of Vbl, an effect that
illustrates the electrogenic contribution of the
Na+-K+-ATPase to Vbl. A summary of
experiments from seven cells originating from one collecting duct and six
collecting tubules with a mean Vbl of -78±4 mV is
shown in Fig. 6B. Upon addition
of ouabain to the bath control solution, Vbl depolarized
by 11±2 mV.
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In six cells, three from collecting tubules and three from collecting ducts the effect of the loop diuretic furosemide on Na+-K+-Cl- cotransport was tested, and was found to have no significant effect on Vbl (Fig. 6C).
These experiments indicate the presence of a Na+-K+-ATPase in the basolateral cell membrane. We found no evidence for the presence of a Na+-K+-Cl- cotransporter, hence, K+ entry into the cell across the basolateral cell membrane may be mediated by the pump.
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Discussion |
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The collecting duct system of amphibians is a heterocellular epithelium
composed of principal and intercalated cells
(Møbjerg et al., 1998).
In this context it should be noted that the Vbl of
collecting duct cells fell into two groups, one being more hyperpolarized than
the other. We do not know if this distribution
(Fig. 2A) reflects two
populations of cells in different functional states, or cells with different
transport characteristics, e.g. principal and intercalated cells. In the
present study it was not possible to distinguish between principal and
intercalated cells at a functional level, as K+ conductances were
similar in all cells. In an ultrastructural investigation of the distal
nephron, Stanton et al. (1984
)
reported that K+ adaptation in Amphiuma means led to an
increase in the basolateral membrane area of principal cells in collecting
tubules. A similar increase was not observed in the intercalated cells. These
data indicate that K+ secretion is limited to the principal cells.
It has been proposed that in the mammalian cortical collecting tubule an
apical H+-K+-ATPase in the luminal membrane of
intercalated cells enables this minority cell type to reabsorb K+
(for a review, see Giebisch,
1998
). In this context it is interesting that
H+-K+-ATPase activity has been demonstrated in the late
distal tubule and collecting tubule from Necturus maculosus and from
Rana ridibunda (see Planelles et
al., 1991
).
We found that the basolateral cell membrane of cells from both collecting
tubules and collecting ducts possessed a large K+ conductance. Our
evidence for the K+ conductance is relatively straightforward and
consistent with previous published studies on the collecting tubule of
Amphiuma sp. (see Horisberger et
al., 1987; Hunter et al.,
1987
). Raising the concentration of K+ in the bath
solution resulted in a reversible depolarization of Vbl.
The addition of millimolar concentrations of the K+ channel
inhibitor Ba2+ to the bath solution inhibited the basolateral
K+ conductance. Assuming that the intracellular K+
activity of the cells in the collecting duct system of Bufo bufo is
in the same range (56 mmoll-1) as the one measured with
ion-sensitive microelectrodes in the collecting tubule of Amphiuma by
Horisberger and Giebisch
(1988
), the equilibrium
potential (given by the Nernst equation), when using the ion activities for
K+ in control solution and in high [K+] solution, would
be approximately -83 mV and -35 mV, respectively. These values are in
agreement with the results presented for the most hyperpolarized cells in
Fig. 3B and indicate that
K+ is the main contributor to the total cell conductance.
This study provides the first evidence for K+ conductance in the apical cell membrane of cells from the collecting duct system of an amphibian. Our experiments on the toad show that Vbl depolarized rapidly in cells from both collecting tubules and collecting ducts in response to luminal perfusion with a high [K+] solution (Fig. 3D, Fig. 5A). Changing the luminal K+ concentration had a less pronounced effect on Vbl, indicating that the contribution of the apical K+ conductance to total cell conductance was smaller than that of the basolateral conductance. Ba2+ inhibited the depolarizing effect of the high [K+] solution in the luminal fluid, which confirms the presence of a K+-conducting pathway in the apical cell membrane. The influence of luminal Ba2+ seemed to be determined by the magnitude of the basolateral K+ conductance to Vbl.
A low-conductance K+-selective channel, such as the one
described by Frindt and Palmer (1989) for the apical cell membrane of the rat
cortical collecting tubule and considered to play an essential role in
K+ secretion in this mammalian species
(Palmer, 1999), has not been
found in amphibians. Nevertheless, in recent studies Stoner and Viggiano
(1998
,
1999
,
2000
) used the patch-clamp
technique and described single K+ channel currents in the apical
cell membrane of the collecting tubule of larval Ambystoma tigrinum.
Maxi K+ channels and intermediate-conductance, non-specific cation
channels were active under hyperkalemic conditions, and may therefore be
involved in K+ secretion in K+-adapted salamanders.
Whether these channels contribute to K+ secretion under normal
physiological conditions is unknown. It is also unknown whether the channels
underlie K+ entry into the lumen of the collecting duct system in
Bufo bufo.
In the urine of Bufo bufo we found a large variation in K+ concentration, consistent with the presence of transport mechanisms responsible for K+ reabsorption and secretion in the kidney or bladder epithelium. We propose that under normal physiological conditions, cells of the collecting duct system of B. bufo secrete K+ into the urine in two steps. K+ is actively taken into the cell over the basolateral cell membrane by the Na+-K+-ATPase and a large K+ conductance in the basolateral cell membrane recycles K+ for the pump. Lack of an effect of basally applied furosemide indicates that transport by a Na+-K+-Cl- cotransporter is not significant. K+ channels in the apical cell membrane provide a path by which K+ can diffuse down its electrochemical gradient into the lumen of the tubule.
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Acknowledgments |
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