1 Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029-6574; and 2 Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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Na+ absorption in the renal cortical collecting duct (CCD)
is mediated by apical epithelial Na+ channels (ENaCs). The
CCD is subject to continuous variations in intraluminal flow rate that
we speculate alters hydrostatic pressure, membrane stretch, and shear
stress. Although ENaCs share limited sequence homology with putative
mechanosensitive ion channels in Caenorhabditis elegans,
controversy exists as to whether ENaCs are regulated by
biomechanical forces. We examined the effect of varying the rate of
fluid flow on whole cell Na+ currents
(INa) in oocytes expressing mouse
,
,
-ENaC (mENaC) and on net Na+ absorption in
microperfused rabbit CCDs. Oocytes injected with mENaC but not water
responded to the initiation of superfusate flow (to 4-6 ml/min)
with a reversible threefold stimulation of INa
without a change in reversal potential. The increase in
INa was variable among oocytes. CCDs responded
to a threefold increase in rate of luminal flow with a twofold increase
in the rate of net Na+ absorption. An increase in luminal
viscosity achieved by addition of 5% dextran to the luminal perfusate
did not alter the rate of net Na+ absorption, suggesting
that shear stress does not influence Na+ transport in the
CCD. In sum, our data suggest that flow stimulation of ENaC activity
and Na+ absorption is mediated by an increase in
hydrostatic pressure and/or membrane stretch. We propose that
intraluminal flow rate may be an important regulator of channel
activity in the CCD.
epithelial sodium channel; ROMK; collecting duct; transepithelial transport; oocyte
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INTRODUCTION |
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THE MAMMALIAN KIDNEY FILTERS large quantities of Na+ on a daily basis. Filtered Na+ is almost entirely reabsorbed within the nephron by a variety of distinct Na+-selective transport proteins expressed in the apical plasma membranes of renal tubular epithelial cells. The mammalian cortical collecting duct (CCD) is a major regulatory site of renal Na+ reabsorption (36, 46, 48, 53). Transcellular Na+ transport in this segment requires Na+ entry across the apical membrane of principal cells through amiloride-sensitive epithelial Na+ channels (ENaCs) (40, 45, 47) and its electrogenic extrusion at the basolateral membrane by the Na+-K+ pump. K+, which accumulates in high concentration within the cell due to Na+-K+-ATPase-mediated basolateral exchange of K+ for Na+, passively diffuses from the cell into the tubular fluid through apical secretory K+ (SK) channels (17, 20, 60). The magnitude of K+ secretion is determined by its electrochemical gradient and by the permeability of the membrane to K+. ROMK, a member of the family of inwardly rectifying K+ channels that is expressed in the mammalian CCD (7, 23, 64, 65), is considered to represent the major functional subunit of the SK channel. This cell model predicts that transepithelial Na+ absorption in the CCD is a major determinant of the rate of K+ secretion.
ENaC, initially cloned from rat colon, exists as a multimeric complex
comprised of homologous -,
-, and
-subunits (6, 8). ENaCs are localized to the apical membrane of epithelial cells not only of colon and distal nephron but also of airway and ducts
of several secretory glands, where they constitute the rate-limiting
step for Na+ reabsorption (45). Their activity
is regulated by a variety of intracellular ions (Na+,
Ca2+, H+) (4, 9, 41); selected
kinases (protein kinases A and C, and sgk) (3, 11, 27);
the ubiquitin ligase NEDD4 (21, 51); extracellular factors
(aldosterone, arginine vasopressin, insulin, proteases) (31, 33,
37, 59); and other integral membrane proteins (i.e., CFTR)
(44, 51, 55). The observation that ENaC subunits show
structural homology to a family of Caenorhabditis elegans
degenerin proteins, including mec-4, mec-10, and
deg-1, proposed to form mechanosensory ion channels
(18, 24, 25), has led to the speculation that ENaC is
sensitive to membrane stretch.
Studies directed at examining the mechanosensitivity of ENaC have produced conflicting results. Palmer and Frindt (42) showed that negative hydrostatic pressure applied to the patch-clamp pipette led to a reversible increase in open probability (Po) of native apical Na+ channels in rat CCD in 6 of 22 patches. However, the majority of patches (15 of 22) showed no response to an increase in transmembrane pressure. These investigators suggested that the inconsistent response of ENaC to stretch and/or pressure reflected variability in the mechanical deformation of the apical membrane within the tip of the pipette.
The mechanosensitivity of ENaC has also been examined in nonepithelial
expression systems. ENaC -subunits expressed in mouse fibroblasts
were activated in response to increases in negative hydrostatic
pressure applied to the patch-clamp pipettes (32).
,
,
-ENaC expressed in lipid bilayers was activated when a
hydrostatic pressure gradient was applied across the bilayer
(26). Xenopus laevis oocytes
expressing
,
,
-ENaC responded to cell swelling with either no
change (5) or a decrease (29) in
Na+ conductance and responded to cell shrinkage with an
increase (29) or decrease (5) in
Na+ conductance. Achard et al. (1)
demonstrated in human B lymphocytes that a modest increase in the
hydrostatic pressure of the solution bathing the cells activated an
amiloride-sensitive Na+ channel, a response requiring an
intact cytoskeleton. Furthermore, in that study, membrane stretch
altered the amiloride sensitivity, cation selectivity, and
inward-rectifying behavior of this channel when studied in the whole
cell, patch-clamp configuration. Given the differences among the
experimental systems and protocols used to elicit changes in
transmembrane pressure, and the presumed variability in relative
abundance of ENaC subunits and associated proteins expressed in each
system, it is not unexpected that discrepant results have been reported.
The physiological relevance of the mechanosensitivity of ENaC becomes clearly apparent in the mammalian CCD, a nephron segment subject to continuous variations in rates of tubular flow. We examined the effect of increases in the rate of fluid flow on exogenous ENaC channels expressed in X. laevis oocytes and endogenous amiloride-sensitive Na+ channels in rabbit CCDs. Our results suggest that ENaC is a flow-regulated ion channel.
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METHODS |
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Reagents. All chemicals were from Sigma (St. Louis, MO) unless stated otherwise.
Oocyte expression.
We previously cloned and characterized mouse ,
,
-ENaC (mENaC)
cDNAs (2). cDNA for ROMK1 was a gift from S. Hebert
(Vanderbilt University). cRNAs for wild-type
-,
-, and
-mENaC
subunits were synthesized with T3 RNA polymerase (Ambion, Austin, TX). cRNA for ROMK1 was synthesized with T7 RNA polymerase (Ambion). Stage
V-VI X. laevis oocytes pretreated with 2 mg/ml type IV
collagenase were injected with 4 ng of cRNA of each mENaC subunit or 4 ng of ROMK1 cRNA in 50 nl of H2O. After injection, oocytes
were incubated at 18°C in modified Barth's saline (MBS; Table
1) containing 10 µg/ml sodium
penicillin, 10 µg/ml streptomycin sulfate, and 100 µg/ml gentamicin
sulfate, pH 7.2.
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Measurement of cation transport in microperfused CCDs. Adult female New Zealand White rabbits were obtained from Charles River (Quebec, ON) or Covance (Denver, PA). A single CCD was microdissected from each animal, transferred immediately to a temperature and O2-CO2-controlled specimen chamber, and mounted on concentric glass pipettes, as previously described (46). Segments were perfused and bathed at 37°C in either Burg's solution or, to measure transport in the presence of Ba2+, a HEPES-buffered solution (61) (Table 1). Both solutions were adjusted to 290 ± 2 mosmol/kgH2O. Because there were no transepithelial osmotic gradients, water transport was assumed to be zero (46). Burg's solution was bubbled with and then continuously suffused with 95% O2-5% CO2 to maintain pH at 7.4 at 37°C. To examine whether differences in luminal Na+ concentration and/or flow affect the magnitude of Na+ backflux, the rate of "bath-to-lumen" Na+ transport was measured in several CCDs perfused with a 0-Na+ solution (Table 1; 260 mosmol/kgH2O) (52) containing 0.1 mM amiloride to inhibit the lumen-to-bath ENaC-mediated Na+ absorptive flux. In all microperfusion studies, samples of tubular fluid were collected under water-saturated light mineral oil by timed filling of a precalibrated 20-nl volumetric constriction pipette. Flow rate was varied by adjusting the height of the perfusate reservoir.
To study flow-dependent transport, the sequence of flow rates was randomized within each group of tubules to minimize any bias induced by time-dependent changes in transport. In general, three to four timed collections of tubular fluid were made at each of two to three flow rates in a given segment. The concentrations of Na+ and K+ in the samples of collected tubular fluid were measured by helium glow photometry, and the mean rate of ion transport (in pmol · minStatistics. Data are expressed as means ± SE; n equals the number of oocytes or tubules. Significant differences were determined by paired t-test, analysis of variance, or linear regression analysis, as appropriate. Significance was asserted if P < 0.05.
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RESULTS |
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Flow activates whole cell Na+ currents in oocytes
expressing ,
,
-mENaC.
X. laevis oocytes injected with
-,
-, and
-mENaC
cRNA were examined by using a two-electrode voltage clamp at
100 mV
in the presence and absence of perfusion with the Na+
gluconate solution. In the absence of perfusion (i.e., no flow), the
whole cell Na+ current (INa) was
10.0 ± 1.6 µA (n = 55; Fig.
1, A and C). Initiating bath flow from 0 (no flow) to the range of 4-6 ml/min led to a 3.3 ± 0.4-fold increase in amiloride-sensitive
INa (n = 55; P < 0.001; Fig. 1, A and C). Amiloride-sensitive
Na+ currents were not observed in water-injected oocytes
either in the absence or presence of flow (n = 10; Fig.
1B). The maximal increase in INa in
response to flow was quite variable (Fig. 1D), and in 12 of
55 oocytes the increase was <40%. Given the variability in the flow
response, it was difficult to establish a dose-response relationship
(i.e., flow rate vs. INa) with the oocyte
expression system. Flow-mediated increases in
INa were observed within 1 min after initiation
of oocyte perfusion and reached a plateau within 3-5 min (Fig.
1E). Flow activation of INa was
reversible, as INa fell when the flow was
stopped. Flow activation of INa was not
associated with a significant change in the resting membrane potential
of the oocyte (Fig. 1F). The reversal potential of oocytes expressing mENaC was 2.1 ± 0.9 mV in the absence of flow and
0.3 ± 0.9 mV in the presence of flow (n = 20;
P = 0.18). These data suggest that the Na+
concentration gradient across the oocytes was not appreciably altered
by flow.
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Flow activates Na+ absorption in rabbit CCD.
To examine the effect of flow rate on net Na+ absorption in
the rabbit CCD, single CCDs were perfused at increasing flow rates and
the rates of net Na+ absorption were measured. The
relationship between flow rate and net Na+ absorption in
the CCD perfused in the presence of Burg's solution is shown in Fig.
3. The rate of net Na+ absorption
increased significantly as flow rate was increased from 0.4 to 3 nl · min1 · mm
1
(r = 0.97; P < 0.05 by linear
regression analysis), the physiological range of flow rates reported
for the rabbit distal nephron (12). Vte remained unchanged as flow rate was
increased (P = 0.26; Table 2). As reported previously by ourselves
(46) and others (15, 22, 35, 52), net
K+ secretion in the CCD was also stimulated by increasing
flow rate (Table 2). In agreement with previous observations (38,
54), 0.1 mM amiloride added to the luminal perfusate
(n = 4) significantly inhibited net Na+
absorption compared with transport rates measured at comparable flow
rates in the absence of the inhibitor (P < 0.03; Fig.
3).
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DISCUSSION |
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Flow rates within the distal nephron, including the cortical collecting duct, increase in response to expansion of the extracellular fluid volume or administration of diuretics and fall in response to volume depletion (20). These clinical observations led us to speculate that flow-induced changes in hydrostatic pressure, cell membrane stretch, or fluid shear stress might directly or indirectly regulate activity of apical ion channels in this nephron segment. Given the importance of the collecting duct in Na+ absorption and the evidence suggesting that ENaC may be a mechanosensitive ion channel, we sought to examine whether ENaC activity and Na+ transport are regulated by fluid flow rate.
Our results show that amiloride-sensitive Na+ currents in X. laevis oocytes expressing ENaCs are flow activated (Fig. 1) in a reversible manner. The response to flow was quite variable as we observed little or no response to changes in flow rates in some experiments (Fig. 1D). We speculate that this variability in response may be due, at least in part, to the high baseline variability in ENaC Po that has been previously reported in rat principal cells (42) and M-1 mouse collecting duct cells (10).
Mechanosensitive channels in the worm C. elegans are tethered to both extracellular and intracellular proteins (56), and these interactions are thought to be required for mechanotransduction. Oocytes expressing ENaCs are surrounded by a vitelline membrane that could serve a similar purpose in the variable mechanosensory response of ENaC expressed in oocytes. We have observed that oocytes stripped of their vitelline membrane are too fragile for perfusion experiments to examine whether there is retention or loss of flow activation of ENaC.
In microperfused rabbit CCDs, an increase in tubular fluid flow rate
within the physiological range is associated with an increase in
amiloride-inhibitable net transepithelial Na+ absorption
(Figs. 3 and 4). This flow-induced response was not due to a reduction
in Na+ backflux into the lumen at high flow rates.
Furthermore, flow-stimulated Na+ absorption can be
dissociated from net K+ secretion and is not accompanied by
an increase in Vte. The absence of flow-induced
change in Vte, even under conditions where
apical K+ secretion was inhibited, may reflect an increase
in the paracellular permeability to Cl, leading to
movement of negative charge out of the lumen.
In contrast to the more than threefold increase in amiloride-sensitive Na+ currents induced by flow over oocytes expressing ENaC, flow led to only a modest increase in K+ currents in oocytes injected with ROMK cRNA (Fig. 2). The low sensitivity of ROMK to flow is compatible with parallel observations in isolated perfused CCDs showing that flow-induced stimulation of net K+ secretion in isolated perfused rabbit CCDs is blocked by TEA (63), an inhibitor of maxi-K but not SK or ROMK channels (16, 23, 60, 64). These data suggest that flow-dependent K+ secretion is mediated by a maxi-K channel whereas baseline K+ secretion is mediated by the SK/ROMK channel.
It is well established that NaHCO3 reabsorption in the proximal tubule increases in response to an increase in axial flow rate due to flow-dependent stimulation of Na+/H+ antiporter activity (43). Perfusion rate has also been shown to modulate Na+/H+ exchange and the rate of H+ secretion in cultured opossum kidney cells (19). Flow dependence of solute reabsorption in the proximal tubule, with its apical brush-border membrane, has been attributed to flow rate-induced alterations in concentration gradients in the vicinity of the apical membrane. However, using a mathematical modeling approach, Krahn and Weinstein (34) were unable to demonstrate that gradients accumulate in an unstirred layer at the apical membrane of the proximal tubule brush border. We do not consider that an unstirred layer effect contributes significantly to our observations. First, in the oocyte experiments, there was no significant difference between the reversal potential measured at zero and high flow rates. This suggests that the flow-induced increase in INa is not due to changes in the relative Na+ concentration at the external face of the channel; i.e., imposition of high flow did not alter the Na+ concentration in an unstirred layer at the cell membrane. Although the magnitude of unstirred layers in the intact CCD is unknown, we speculate that it is no greater than that in the proximal tubule. Furthermore, the maximal difference (20 mM; Table 2) between Na+ concentrations of perfused (~140 mM) and collected tubular fluids indicates that it is likely that Na+ concentrations at the CCD plasma membrane were always well in excess of the Michaelis-Menten constant of ENaC for Na+, recently reported to be 38 mM (30).
Mechanical forces regulate cell function in a variety of tissues but have been particularly well studied in endothelial cells (13, 57). Because of their location within the vasculature, endothelial cells experience three types of mechanical forces: hydrostatic pressure, circumferential stretch or tension, and fluid shear stress generated by the frictional force of blood (13, 57). Of these, shear stress may be particularly important because of its effects on cytoskeletal remodeling, release of growth factors and vasoactive substances, and changes in gene expression, cell metabolism, and cell morphology (13, 57). Although the signaling pathways by which physical forces are transduced into biochemical signals are actively under investigation, it is apparent that the mechanosensitive ion channel represents a common mechanism by which a variety of cells sense changes in mechanical stimuli. Several different mechanosensitive ion channels have been identified in endothelial cells, including a shear-responsive voltage-gated Na+ channel (58) and K+ channel and a stretch-activated Ca2+ channel (14, 28).
We propose that mechanical forces regulate the function of renal tubular cells, as they do in endothelial cells. Our observation that a 5.5-fold increase in luminal viscosity did not alter the rate of net Na+ absorption in CCDs perfused at a constant flow rate (Fig. 5) suggests that flow-induced stimulation of Na+ transport is not due to an alteration in the frictional forces (shear stress) to which the apical surfaces of epithelial cells were exposed. Whether flow stimulation of ENaC activity and transepithelial Na+ absorption are due to an increase in hydrostatic pressure and/or membrane stretch remains to be explored. Also not addressed by the present studies is whether this represents a direct effect on the channel protein or an indirect effect transduced by an as yet unidentified signaling pathway. Investigation of these questions represents an important goal toward understanding the regulation of renal epithelial cell function in the context of the physical forces that prevail in vivo.
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ACKNOWLEDGEMENTS |
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The authors thank Beth Zavilowitz for technical support.
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FOOTNOTES |
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* L. M. Satlin and S. Sheng contributed equally to this study.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-38470 (L. M. Satlin) and DK-51391 (T. R. Kleyman), an American Heart Association Grant-in-Aid (L. M. Satlin), and the Department of Veterans Affairs. S. Sheng is the recipient of a postdoctoral fellowship award from the Cystic Fibrosis Foundation. Abstracts of this work have been presented at the Annual Meetings of the American Society of Nephrology in Philadelphia, PA, in October 1998 and Society for Pediatric Research in San Francisco, CA, in May, 1999.
Address for reprint requests and other correspondence: L. M. Satlin, Box 1664, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, N.Y. 10029-6574 (E-mail: lisa.satlin{at}mssm.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 November 2000; accepted in final form 13 February 2001.
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