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*Physiologisches Institut, Albert-Ludwigs-Universität, Hermann-Herder-Straße 7, 79104 Freiburg, Germany; Department of Physiology, Sungkyunkwan University School of Medicine, Suwon 440-746, South Korea.
Correspondence to Jens Leipziger, Institute of Physiology, Ole Worms Allé 160, 8000 Aarhus C, Denmark. Phone: +45-89-422800; Fax: +45-86-129065; E-mail: leipziger{at}fi.au.dk
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cm2; and equivalent short-circuit current (Isc), -261.0 ± 25.1 µA/cm2 (n = 45). Amiloride (10 µM) almost completely inhibited Isc to -3.9 ± 3.8 µA/cm2 (n = 10). Luminal ATP (100 µM) reduced Vte from -16.5 ± 2.1 to -12.5 ± 1.93 and increased Rte from 113.1 ± 16.2 to 123.8 ± 16.7 cm2, which resulted in a 31.7% inhibition of amiloride-sensitive Isc (n = 12). Similarly, luminal UTP reversibly reduced Vte from -22.0 ± 2.1 to -13.6 ± 2.1 mV and increased Rte from 48.4 ± 5.3 to 59.2 ± 7.1 cm2, which resulted in 49.1% inhibition of Na+ absorption (n = 6). In parallel, luminal ATP and UTP elevated [Ca2+]i in CCD, increasing the fura-2 ratio by 2.7 ± 0.7 and 4.0 ± 1.2, respectively. Basolateral ATP and UTP (100 µM) also inhibited amiloride-sensitive Isc by 21.8 (n = 14) and 20.1% (n = 8), respectively. Inhibition of luminal nucleotide-induced [Ca2+]i increase by Ca2+ store depletion with cyclopiazonic acid (3 µM) did not affect nucleotide-mediated inhibition of Na+ transport (n = 7). No evidence indicated the activation of a luminal Ca2+-activated Cl- conductance, a phenomenon previously shown in M-1 CCD cells (J Physiol 524: 77–99, 2000). In essence, these data indicate that luminal ATP and UTP, most likely via P2Y2 receptors, mediate inhibition of amiloride-sensitive Isc in perfused mouse CCD. This inhibition appears to occurs independently of an increase of cytosolic Ca2+. |
Introduction |
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Freshly isolated mammalian kidney tubules express a number of different P2 receptors, apparently along the entire nephron (12). Functional expression of basolateral P2Y receptors could be demonstrated, e.g., in mouse loop of Henle (13), rabbit cortical collecting duct (CCD) (14), or rat inner medullary collecting duct (15–17). In addition, in our earlier article (18), we identified a luminal P2Y2 receptor coupled to an increase of [Ca2+]i in the isolated perfused mouse CCD. Using a confocal microscope, we could localize the luminal ATP-induced [Ca2+]i elevation to principal cells. The data indicated that the P2Y2 receptor subtype was the only P2 receptor expressed in the luminal membrane of mouse principal cells. Our report is in close agreement with a recent immunohistochemistry study that demonstrated the expression of P2Y2 receptors in the basolateral, which was even more pronounced in the luminal membrane of rat inner medullary collecting duct principal cells (15). Studies that have focused on functional consequences of P2 receptor activation in intact kidney tubules are scarce. Basolateral ATP has been shown to inhibit the antidiruetic hormone (ADH)-induced increase in H2O permeability in rat inner medullary collecting duct (16) and rabbit CCD (14). In rat inner medullary collecting duct, the inhibition of H2O transport was attributed to a P2 receptor-mediated decrease of cyclic ATP (16). A number of investigators, including ourselves, have used different cell lines derived from renal tubular cells in an Ussing chamber set-up to investigate the effect of basolateral and luminal nucleotides on electrogenic ion transport. Two key findings were observed, namely that luminal and basolateral nucleotides activate Cl- secretion (19–21) and inhibit amiloride-sensitive Na+ absorption (19,20,22). The Cl- secretion, e.g., observed in M-1 CCD cells, is activated by an increase in [Ca2+]i (20), whereas the inhibition of Na+ transport appears to be regulated [Ca2+]i-independently (22,23).
In this follow-up work, we investigate the effect of luminal and basolateral ATP and uridine triphosphate (UTP) on electrogenic Na+ absorption in perfused mouse CCD. To this end, mice were treated with a low Na+ diet for 10 to 20 d. We were also interested in whether the intact CCD is equipped with luminal Ca2+-activated Cl- channels. In M-1 CCD cells, this was reported in our study elsewhere (20).
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Materials and Methods |
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Isolated Tubule Perfusion and Measurement of Transepithelial Electrical Parameters
Isolated tubules were perfused by use of a system of concentric glass pipettes previously used and developed by R. F. Greger and W. Hampel (24). To quantify electrogenic Na+ transport, Ohm’s law was applied:
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| (1) |
where Isc is the equivalent short-circuit current, Vte the transepithelial voltage, and Rte the transepithelial resistance. Vte was measured continuously on both ends of the perfused CCD. The system for recording was symmetric, so that no corrections due to superimposed liquid junction potentials were necessary when identical solutions were present on both sides of the epithelium. The determination of Rte was performed by use of the cable equation (equation 3). A detailed description of the quantitative estimation of electrogenic ion transport in kidney tubules is given in publications elsewhere (25–27). Rte ( cm2) was calculated from the input resistance RINP (), the length constant 
(µm), the length of the tubule segment L (µm), and the resistivity 
( cm) of the perfusate. To obtain RINP and rectangular current pulses (I0, 50 nA; pulse width 800 ms every 3 s), were injected through one channel of the perfusion pipette. The corresponding voltage deflections 
V0 at the perfusion side and VL at the collection side were measured by two voltmeters (Keithley Instruments, Cleveland, OH). RINP is defined as V0/I0. was obtained from the exponential equation 2, as given by Helman et al. (25):
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The length and the inner radius r (µm) of the tubule were measured by a size-calibrated transmission image. Previously obtained resistivity values were used. For the calculation of Rte, the following equation was used:
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| (3) |
A slightly different approach was taken for long tubule segments in which no accurate value of VL could be obtained. Herein, equation 4 was used to determine (27):
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| (4) |
Digital Video Imaging
The setup consisted of an inverted microscope (Axiovert 100 TV; Zeiss, Jena, Germany) with a 40x objective (Fluar 40x, 1.3 oil; Zeiss, Jena, Germany), a monochromator (Till Photonics, Planegg, Germany), and a GEN 3 intensified CCD camera (ICCD 350; Videoscope, Dulles, VA). Image acquisition and data analysis were performed with the software package Metamorph/Metafluor (Universal Imaging, West Chester, PA). Freshly dissected CCD were mounted into the perfusion system previously built and used in this laboratory (24). Measurement of [Ca2+]i was performed with the Ca2+-dye fura-2. Tubules were incubated in 20 µM basolateral fura-2/AM for 15 min at room temperature in Ringer’s solution to which 1.6 µM pluronic F127 had been added. Pluronic F127 is a surfactant polyol that helps solubilize water-insoluble dyes like fura-2/AM. As a measure of [Ca2+]I, the fluorescence emission ratio at 345/380 nm excitation was used. In each experiment, the fluorescence signal was recorded from the entire tubule. During the dye-loading period, the tubule was continuously perfused from the luminal side. The experiment was started 5 min after washout of extracellular dye. To demonstrate successful luminal agonists perfusion, we added 10 µM lucifer yellow (LY, excitation 430 nm and emission >500 nm) to the luminal perfusate. LY is not excitable with the two fura-2 wavelengths (345 and 380 nm), and fura-2 is not excited with 430 nm light. Thus, the two fluorescence signals could be observed without significant cross-contamination. Images were recorded sequentially after 345, 380, and 430 nm excitation. One fura-2 ratio and the LY fluorescence were recorded every 2 s, respectively.
Solutions and Chemicals
Pluronic F127, LY, and fura-2/AM were obtained from Molecular Probes (Eugene, OR). All other chemicals were of the highest grade of purity available and were obtained from Sigma (Deisenhofen, Germany) and Merck (Darmstadt, Germany). The experiments of continuous basolateral and luminal perfusion of kidney tubules were performed with the following solutions (in mM): 145 NaCl, 1 MgCl2, 1.3 Ca-gluconate, 5 D-glucose, 0.4 KH2PO4, and 1.6 K2HPO4. A few experiments were also performed in a HCO3-buffered solution (in mM): 120 NaCl, 1 MgCl2, 1.3 Ca-gluconate, 5 D-glucose, 0.4 KH2PO4, 1.6 K2HPO4, and 25 NaHCO3 gassed with 95% O2/5%CO2. All solutions were titrated to pH 7.4. Kidney tubules were dissected in F12 (HAM) medium (Life Technologies, Karlsruhe, Germany) to which 5 mM glycin, 10 µM amiloride, and 1 mg/ml bovine serum albumin had been added.
Statistical Analyses
The data shown are either original traces or mean values ± SEM (n), where n refers to the number of experiments. A paired t test was used to compare mean values within one experimental series. A P < 0.05 was accepted to indicate statistical significance.
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cm2; and calculated equivalent Isc, -261.0 ± 25.1 µA/cm2 (n = 45). In a series of 10 CCD, we investigated the effect of 10 µM luminal amiloride (Figure 1). Luminal amiloride reversibly decreased lumen-negative Vte from -16.3 ± 2.5 to -0.3 ± 0.5 mV and increased Rte from 113.3 ± 19.3 to 141.2 ± 28.6 cm2, which resulted in nearly complete inhibition of Isc from -173.5 ± 36.2 to -3.9 ± 3.8 µA/cm2. These results indicate that a low-Na+ diet substantially activates electrogenic Na+ absorption in CCD of mice, which is completely inhibitable by luminal amiloride.
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Vte induced by injection of a 50 nA current. The middle panel depicts the [Ca2+]i signal of the entire tubule measured as the fura-2 345/380 nm fluorescence ratio. The bottom panel depicts the measurement of LY fluorescence intensity as indicator for luminal perfusion. In this CCD, resting Vte was close to -17 mV. The addition of luminal ATP (100 µM) slowly, within 5 to 6 min, reduced Vte to -9 mV. Subsequent addition of luminal amiloride (10 µM) rapidly reduced Vte further to 2.5 mV. A close inspection of the upper panel also reveals that during perfusion with luminal ATP the Vte deflections increase, reflecting an increase in Rte (from 123.3 to 179.0 cm2). As has been previously demonstrated, luminal ATP also increased [Ca2+]I, with an initial peak leveling off toward the baseline during the following 5 to 7 min of ATP administration. It is noteworthy that no close temporal correlation could be observed between the stimulated [Ca2+]i transient and the reduction of Vte or the increase of Rte. The luminal amiloride solution did not contain LY, which explains the wash-out of the luminal "perfusion marker" during the effect of amiloride. Our previous study (18) strongly suggested that principal cells of mouse CCD express a luminal P2Y2 receptor. Thus, it must be expected that luminal UTP, the other known ligand of this receptor, should have similar effects. A single experiment is depicted in Figure 2B. The addition of luminal UTP (100 µM) reversibly reduced resting Vte from -29 to -17 mV and increased Rte, as is seen from an increase in Vte deflections (from 69.9 to 91.6 m2). Luminal UTP also elevated [Ca2+]i in a biphasic manner, with an initial peak and a secondary plateau. Thus, luminal UTP induces similar effects as luminal ATP. Again, no close temporal correlation was observed between the [Ca2+]i elevation and the effect on Vte and Rte. In Figure 3, the entire experimental series with luminal ATP is summarized. Luminal ATP reversibly reduced Vte from -16.5 ± 2.1 to -12.5 ± 1.9 mV and increased Rte from 113.1 ± 16.2 to 123.8 ± 16.7 cm2 (n = 12). This led to a reduction of Isc from -201.3 ± 46.0 to -140.8 ± 35.0 µA/cm2, 31.7% inhibition of Na+ absorption. Most experiments were performed as shown in Figure 2A, with direct administration of luminal amiloride following that of luminal ATP. Often, luminal washout lasted too long to demonstrate full reversibility of the ATP effect. In the UTP series, however, as also shown in Figure 2B, reversibility of the effect is shown unequivocally. This is also reflected in the mean values of the UTP experiments. Luminal UTP reduced Vte from -22.0 ± 2.1 to -13.6 ± 2.1, and Vte returned back to -20.5 ± 2.7 mV after UTP washout. UTP also reversibly increased Rte from a resting value of 48.4 ± 5.3 to 59.2 ± 7.1 cm2. This resulted in a reduction of amiloride-sensitive short circuit current from -478.3 ± 67.3 to -248.3 ± 49.4 and after washout of UTP back to -426.8 ± 82.1 µA/cm2 (n = 6). Altogether, UTP inhibited 49.1% of the entire Na+ transport.
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Vte and the lower panel that of [Ca2+]i. The addition of basolateral ATP (100 µM) reversibly reduced Vte from a resting value of -26 to -24 mV. Simultaneously, Rte increased, as reflected by an increase of Vte deflections (from 58.0 to 68.9 cm2). The summary of all data with 100 µM basolateral ATP is shown in Figure 5A. Vte was reversibly reduced from -13.6 ± 2.5 to -12.0 ± 2.3 mV, and Rte increased from a resting value of 56.5 ± 8.0 to 63.5 ± 9.6 cm2. This resulted in small but significant reduction of amiloride-sensitive Isc from -272.8 ± 53.4 to -210.6 ± 42.1 and back to -261.2 ± 47.9 µA/cm2 (n = 14). Altogether, the basolateral ATP-=mediated inhibition of Na+ absorption amounted to 21.8%. We also used basolateral UTP, which demonstrated effects similar to those described for basolateral ATP. Figure 5B summarizes the entire series of basolateral nucleotides on Na+ transport. Basolateral UTP and ATP inhibited Isc with an inhibitory concentration50 value of 1.24 and 3.22 µM, respectively, with a similar maximal inhibition of Na+ absorption at 100 µM.
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Vte deflections, again indicating an increase in Rte (from 86.9 to 98.2 cm2). In summary, luminal ATP under CPA reduced Vte from -15.0 ± 1.8 to -10.2 ± 1.5 and back to -13.4 ± 3.5 mV, Rte increased from 78.0 ± 13.9 to 89.9 ± 17.8 cm2, and Isc decreased from -226.6 ± 42.3 to -134.6 ± 27.3 µA/cm2, amounting to 37% inhibition of Na+ absorption. Importantly, under these conditions, luminal ATP did not increase but rather reduced [Ca2+]i by 0.6 ± 0.3 fura-2 ratio units (n = 7). These results suggest that the observed inhibition of Na+ absorption possibly occurs independently from an increase of [Ca2+]i. In an attempt to investigate the role of [Ca2+]i further, we also tried BAPTA-AM (50 or 100 µM for 30 min) to buffer the ATP-induced [Ca2+]i increase. Under BAPTA-AM, the initial ATP-induced (100 µM) [Ca2+]i peak was completely inhibited; however, a Ca2+ influx was stimulated, which led to a significant [Ca2+]i increase that extended the duration of either luminal or basolateral ATP application by some 5 min (fura-2 ratio increase 0.67 ± 0.48, n = 4). More importantly, BAPTA-AM incubation reduced Vte from -15.8 ± 6.8 mV to -4.8 ± 3.5 mV (n = 5), rendering the further investigation of Na+ transport almost impossible.
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Regulation of Na+ Transport in the CCD
The collecting duct serves to finely tune the urinary Na+ excretion. The absorptive process is dependent on constitutively open epithelial sodium channel (ENaC) channels in the luminal membrane of principal cells. Na+ is transported transcellularly, enters the cytosol via a steep electrochemical driving force over the apical membrane, and is subsequently pumped into the basolateral space via the Na+/K+ ATPase. Principal cells are the target of aldosterone- and ADH-mediated activation of Na+ transport (29). Na+ absorption is furthermore under the modulation of a variety of different agonists (30). For example, a number of [Ca2+]i elevating agonists like PGE2 or acetylcholine were shown to inhibit electrogenic Na+ transport in rabbit CCD (31,32). More recently, a number of studies also identified different P2 in renal epithelia (16,17,33). A unique phenomenon of many epithelial cells, including the distal nephron, is the expression of P2 receptors in both the luminal and basolateral membrane (9,18).
P2Y Receptor Identification in Mouse CCD
Despite the fact that numerous P2 receptors have been described in transporting epithelia (34), the most prominent luminal subtype is the P2Y2 receptor, expressed, e.g., in bronchial or colonic mucosal tissue (1,2). Similarly, in our earlier article, we presented evidence that the luminal nucleotide-mediated increase of [Ca2+]i in mouse CCD principal cells occurs via a P2Y2 receptor. The conclusion was based on an extensive pharmacologic screening with a large number of different P2 receptor agonists. We currently believe that the P2Y2 receptor is the only P2 receptor expressed in the luminal membrane of mouse CCD principal cells. A number of additional arguments strengthen this conclusion. Our own data in M-1 cells identifies specific mRNA for this receptor subtype and shows a typical pharmacology, with UTP being as potent as ATP (20). Even more conclusive is a recent study in rat inner medullary collecting duct that used a specific antibody that localized this receptor predominantly to the luminal membrane (15). It needs, however, to be considered that pharmacologic evidence with UTP and ATP being of similar potency would also allow the P2Y4 receptor to be a possible candidate (6). Yet no other evidence indicates that the latter receptor is involved.
Mechanism of ATP-Mediated Inhibition of Na+ Transport
In general, the simultaneous function of luminal ENaC channels, the basolateral Na+/K+ ATPase, and a basolateral K+ conductance is required to permit the transcellular movement of Na+ in the distal nephron. Thus the inhibition of any of these transporting modules could account for the observed ATP effect. Our study does not allow us to conclude which of the relevant membrane transport molecules was the target of regulation. An interesting recent cell attached patch-clamp study investigated the effect of P2Y2 receptor stimulation on small-conductance K+ channels (renal outer medulla K+ channel [ROMK]-type) in the luminal membrane of mouse CCD principal cells. Luminal ATP reversibly inhibited 
90% of constitutively open ROMK channels (35). An ATP-mediated closure of ROMK channels will inhibit electrogenic Na+ transport, because this would reduce the steep electrochemical gradient over the luminal membrane necessary for the entry of Na+.
However, if ATP as the sole mechanism would close luminal K+ channels, the transepithelial measurement should have increased to even more lumen-negative Vte values. The opposite was the case—namely, ATP decreased Vte. Thus, because Rte increased, additional ion conductances must have been inhibited with luminal ATP. One attractive hypothesis is that ENaC channels are the target of ATP-mediated inhibition. In other words, luminal ATP would inhibit both ROMK and ENaC channels, with the latter being the dominant effect, to explain the Vte measurements. In this context, a single-channel patch-clamp study in A6 cells (36) suggested that luminal ATP via P2Y receptors inhibits ENaC channels. Also, our previous M-1 work (23) would suggest that ENaC is the target of extracellular ATP regulation.
Is [Ca2+]i Required for the ATP-Mediated Inhibition of Na+ Absorption in CCD?
Over the past two decades, numerous studies that have used microperfused collecting ducts have established that [Ca2+]I-elevating agonists like PGE2 or acetylcholine inhibit Na+ transport (32,37). Mostly, rabbit collecting ducts have been used, and numerous reports support the involvement of a cytosolic [Ca2+]i elevation mediating the inhibition of Na+ transport (32,37,38). Downstream of the [Ca2+]i signal, protein kinase C (PKC) activation has been suggested to mediate the inhibitory effect on Na+ transport (39). Thus, in rabbit tissue, current evidence draws a homogeneous picture of these initial signaling events. In contrast, in rodents like rats, intracellular Ca2+ and PKC do not seem to be involved as inhibitory regulators of electrogenic Na+ absorption (40).
Three arguments would suggest that [Ca2+]i is not a crucial regulator of ATP-mediated Na+ transport inhibition in mice. (1) Under microsomal Ca2+-ATPase inhibition, the ATP-mediated [Ca2+]i increase was obliterated, and the nucleotide under these conditions even decreased [Ca2+]i. This, however, left the Na+ transport inhibition unaltered. However, because Ca2+ entry and basal [Ca2+]i were elevated under continuous CPA, this interpretation is somewhat weakened. (2) In M-1 cells, a much more robust system to investigate possible signaling mechanisms, more aggressive means to lower cytosolic Ca2+, did not alter the ATP-inhibited Na+ absorption. (3) The use of BAPTA-AM in cultured rabbit connecting tubule cells (22) similarly did not effect ATP/UTP-inhibited Na+ and Ca2+ absorption. Thus, we suggest that, also in mice, an elevation of [Ca2+]i is not a necessary event to mediate the observed inhibition.
The previous work in mouse M-1 cells excluded the involvement of PKC (23). In the intact mouse CCD, we tried to use the PKC activator PMA (100 nM); however, we observed a rapid deterioration of tubule morphology and function. On the basis of the above-mentioned data from M-1 cells, we suggest that the activation of PKC may not be involved in the ATP-mediated inhibition of Na+ transport in the intact nephron.
Basolateral nucleotide-mediated inhibition of ADH-stimulated H2O transport in rat inner medullary collecting duct has been suggested to occur via a P2Y receptor-mediated decrease of cAMP (16). As was mentioned above, Na+ absorption in rodents is activated by agonists like ADH, which is known to elevate cAMP (30). It thus may be the case that luminal P2Y2 receptor stimulation decreases cAMP and thereby inhibits Na+ transport. Our study has not addressed this question.
Hypothetical Model for an Integrated Function of ATP as Signaling Molecule along the Nephron
The source of extracellular ATP, especially on the luminal side of epithelia, is still largely obscure. Numerous articles have indicated that ATP can be released by epithelial cells after cellular swelling and therefore may serve as an autocrine-signaling molecule. Following the arguments presented from bile duct epithelial cells (41), we speculated that ATP could serve a role as stimulator of regulatory volume decrease in renal epithelial cells (18). This hypothesis was especially attractive because the kidney is an organ with an outstandingly high demand for efficient volume regulatory mechanisms. One argument in favor of such a hypothesis was that extracellular ATP in cultured renal and other epithelial cells activated Cl- and K+ channels, a key mechanism to reduce cellular volume. This hypothesis is substantially weakened by the current study. Obviously, in the intact CCD, an ATP-stimulated activation of Cl- and K+ channels does not occur. What other functional relevance may be attributed to P2Y receptors in the nephron? One common denominator is that ATP, via P2Y receptors, inhibits transport. This includes, as we show, Na+ absorption, K+ secretion (35), and H2O transport (16). We therefore speculate that ATP could serve as a protective signal when ischemic episodes occur. Ischemia will eventually lead to cell swelling, a phenomenon known to release ATP from epithelial cells. Released ATP, in turn, would bind to P2 receptors and inhibit energy-consuming transcellular transport. In addition, another interesting line of evidence suggests that extracellular ATP could mediate the stimulation of cell proliferation and regeneration in renal tubular cells (42).
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, significant change between the agonist and postcontrol.
