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【关键词】 nucleotides
Centre for Nephrology and Department of Physiology, Royal Free and University College Medical School, London, United Kingdom
ABSTRACT
In vitro evidence suggests that intraluminal nucleotides, acting on apical P2 receptors, may influence amiloride-sensitive sodium reabsorption in collecting ducts. The present study has assessed this possibility directly in anesthetized rats, by determining the urinary recovery of 22Na relative to that of [14C]inulin (Na/inulin recovery ratio) during in vivo microperfusion of late distal tubules with artificial tubular fluid containing various P2 agonists (all at 1 mM). In animals maintained on a control diet, in which amiloride-sensitive 22Na reabsorption was modest, the poorly hydrolysable, broad-spectrum P2 agonist ATPS had no significant effect on the Na/inulin recovery ratio. In contrast, in rats maintained on a low-sodium diet, in which amiloride-sensitive 22Na reabsorption was considerably enhanced, ATPS caused a significant increase in the Na/inulin recovery ratio (control: 14 ± 3%; ATPS: 28 ± 4%; n = 32 pairs; P < 0.001, paired t-test). No change in the Na/inulin recovery ratio was seen in time controls (13 ± 3 vs. 14 ± 4%; n = 15 pairs). In subsequent experiments in rats maintained on a low-sodium diet, we used more selective agonists in an attempt to identify the receptor subtype responsible for the effect of ATPS. The P2Y1 agonist 2meSADP, the P2Y2/4 agonists Ap4A and Cp4U, and the P2X agonist BzATP were all without significant effect on the Na/inulin recovery ratio. These findings constitute the first in vivo evidence for a functional role for apical P2 receptors in collecting ducts, but the identity of the receptor subtype(s) involved remains elusive.
purinoceptors; in vivo microperfusion; ATPS
AS PURINOCEPTOR RESEARCH proliferates, it is becoming increasingly clear that P2 receptors of both P2X and P2Y subtypes are widely distributed in renal epithelia (1, 23). However, their functional roles are far from established. Until recently, most studies have focused on P2 receptors in the basolateral membrane. Basolateral application of nucleotides has been shown to evoke increases in intracellular calcium concentration in most if not all nephron segments (2, 3), and it is well documented that basolateral P2 receptor stimulation in the collecting duct inhibits vasopressin-stimulated water reabsorption (12, 21).
In recent years, attention has shifted to P2 receptors in the apical domain (17), particularly in the distal nephron. In vitro studies, using immunohistochemical and functional approaches, have indicated the presence of apical P2Y receptors in cortical (8, 18) and medullary (13) collecting duct. Application of ATP to the apical surface of mouse cortical collecting tubule (CCT) in vitro has been shown to increase intracellular Ca2+ concentration (8) and to reduce amiloride-sensitive short-circuit current (16), taken to be an index of amiloride-sensitive epithelial sodium channel (ENaC)-mediated sodium reabsorption. Similar effects have been described in the mouse M1 cell line (7, 26), which in some respects resembles collecting duct principal cells. Pharmacological profiling suggests that the apical P2 receptors mediating these responses are of the P2Y2 subtype (8, 16).
Although the above in vitro findings, in cell lines and perfused tubule segments, are valuable pointers to a potential physiological role for apical P2 receptors, the question of whether luminal nucleotides regulate ion transport in the intact epithelium in vivo has not yet been addressed. In the present study, therefore, we attempted the first direct in vivo assessment of the effect of luminal application of P2 receptor agonists on collecting duct sodium reabsorption. Because of the likelihood that any such effect would be amiloride-sensitive, we also investigated whether the response was influenced by sodium status, used as a physiological means of altering the number of ENaC in the distal nephron (20). In the first instance, we used the poorly hydrolysable ATP analog ATPS, a broad-spectrum P2 agonist. Subsequently, more selective agonists were employed, to try to identify the receptor subtype(s) involved.
METHODS
Male Sprague-Dawley rats were maintained on a normal diet (Na+ content 140 mmol/kg dry weight; n = 12 rats) or a low-sodium diet (Na+ content 13 mmol/kg dry weight; Special Diets Services, Witham, Essex, UK; n = 32 rats) for 78 days. They were then anesthetized with sodium thiopentone (100 mg/kg ip; Link Pharmaceuticals, Horsham, Sussex, UK) and prepared surgically for micropuncture of the left kidney (25). Rats on a normal diet were infused intravenously throughout with NaCl solution (150 mM) at a rate of 5 ml/h. Rats on a low-sodium diet were infused with a mixture of 150 mM NaCl (0.5 ml/h) and 2% glucose solution (4.5 ml/h) during surgery and with a 2% glucose solution alone (5 ml/h) thereafter (30).
A micropipette (tip diameter 78 μm), containing 150 mM NaCl solution plus 0.1% FD&C Blue dye, was inserted into a proximal tubule and a small volume was flushed down the tubule so as to identify a late distal tubular site. A second pipette, containing artificial distal tubular fluid and connected to a Hampel (Neu-Isenberg, Germany) microperfusion pump, was used to perfuse the late distal tubule (nominal perfusion rate 3 nl/min; 46 min). The composition of the artificial tubular fluid was 50 mM NaCl, 10 mM KCl, and 5 mM sodium-free HEPES, plus [14C]inulin (15 μCi/ml; Amersham International, Aylesbury, Bucks, UK), 22Na (25 μCi/ml; Amersham International) and 0.07% FD&C Blue dye. Its pH was 6.50.
To monitor [14C]inulin and 22Na recoveries, urine was collected from the ureter of the microperfused kidney directly into vials containing scintillant (Aquasol 2; Canberra Packard, Pangbourne, Berks, UK) for a total of 20 min after the start of microperfusion. Urine was also collected from the contralateral kidney for the determination of overall sodium excretion (measured by flame photometry; model 543, Instrumentation Laboratory, Warrington, Cheshire, UK).
Each late distal tubule was perfused twice, first with the control perfusate (described above), then with one of the following perfusates: 1) control perfusate (time control experiments), 2) perfusate containing amiloride (1 mM; RBI, Natick, MA), or 3) perfusate containing a P2 receptor agonist, each at a concentration of 1 mM. The agonists were adenosine 5'-O-[3-thiotriphosphate] (ATPS; Sigma, Poole, Dorset, UK), P1,P4-di[adenosine-5']tetraphosphate (Ap4A; Sigma), 2-methylthioADP (2meSADP; Sigma), 2',3'-O-[4-benzoylbenzoyl]ATP (BzATP; Sigma) and P1-[cytidine 5'-],P4-[uridine 5'-]tetraphosphate (Cp4U; Inspire Pharmaceuticals, Durham, NC). The microperfusion pump was calibrated by delivering perfusate directly into scintillation vials for 45 min. The activities of [14C]inulin and 22Na were determined by -scintillation spectroscopy (model 2900 TR; Canberra Packard), and converted to disintegrations per minute to allow for variable quenching. The actual perfusion rate was 3.2 ± 0.1 nl/min (means ± SE, n = 18) for the control perfusions and 3.2 ± 0.2 nl/min (n = 18) for the P2 agonist/amiloride perfusions. Perfusions were only accepted if the recovery of [14C]inulin exceeded 85% of the amount delivered. Recovery of 22Na was then calculated as a percentage of [14C]inulin recovery (Na/inulin recovery ratio).
Results are presented as individual paired values or as means ± SE. Statistical comparisons between the first and second values from paired perfusions were made using Student's paired t-test; a one-tailed test was used for amiloride, and a two-tailed test for all other cases. A value of P < 0.05 was considered to be statistically significant.
RESULTS
Sodium-replete rats. Urine flow rate and sodium excretion (contralateral kidney only) were 28 ± 4 μl/min and 4.0 ± 0.4 μmol/min, respectively. With the control perfusate, 4050% of the 22Na delivered to the late distal tubule was excreted in the urine. In the time control experiments, there was no change in the Na/inulin recovery ratio (Fig. 1A). On addition of ATPS to the perfusate, a slight trend toward an increased 22Na recovery was discernible, but the increase in the Na/inulin recovery ratio did not achieve statistical significance (Fig. 1B). Addition of amiloride to the perfusate, however, caused a significant increase in the Na/inulin recovery ratio (Fig. 1C).
Sodium-restricted rats. In sodium-restricted rats, urine flow rate and sodium excretion (contralateral kidney only) were 33 ± 2 μl/min and 0.04 ± 0.01 μmol/min, respectively. In these animals, the baseline Na/inulin recovery ratio during late distal microperfusion was reduced to less than 20%, demonstrating enhanced sodium reabsorption in the collecting duct. Again, in time control experiments there was no change in 22Na recovery (Fig. 2A), but in these animals ATPS caused a significant reduction in sodium reabsorption, the Na/inulin recovery ratio increasing in 28 of 32 pairs of perfusions (Fig. 2B). Note that n is greater for ATPS perfusions than for other perfusions because the former were used as a "positive control" throughout the subsequent part of the study. As would be anticipated, the response to amiloride was substantially greater in sodium-restricted rats than in sodium-replete animals (Fig. 2C).
In the next stage of the study, we attempted to identify the receptor subtype(s) responsible for the effect of ATPS in sodium-restricted rats. Because in vitro studies had suggested that stimulation of apical P2Y2 or P2Y4 receptors in mouse collecting duct could inhibit sodium reabsorption (16), we used Cp4U, believed to be a selective agonist for P2Y2 and (less potently) P2Y4 receptors, and Ap4A, whose P2Y activity is also limited to the P2Y2/P2Y4 subtypes. Figure 3 shows that luminal application of each of these agonists failed to affect the Na/inulin recovery ratio in vivo. In a few perfusions we also tested the naturally occurring P2Y2/P2Y4 agonist UTP (1 mM). This compound was also ineffective (data not shown), although the rapid metabolism of UTP by tubular nucleotidases (35) limits the significance of this observation.
The possible contribution of P2Y1 receptors was assessed by application of the selective P2Y1 agonist 2meSADP. This was also without significant effect in our preparation (Fig. 4A). Finally, to test for a possible role of P2X receptors, we used BzATP. Here, we saw some indication of a very modest increase in the Na/inulin recovery ratio (Fig. 4B), but this did not achieve statistical significance.
DISCUSSION
The first conclusion to be drawn from this study is that the preparation employed provides a useful model with which to investigate collecting duct sodium reabsorption in vivo. Baseline 22Na reabsorption changed in the predicted direction when animals were given a low-sodium diet, and this augmentation of sodium reabsorption was amiloride sensitive, in accord with the documented increase in the number of apical sodium channels (20).
A growing body of evidence suggests that epithelial sodium transport can be regulated by extracellular nucleotides. As indicated earlier, in vitro studies showed that luminal ATP can reduce amiloride-sensitive short-circuit current across collecting duct cells, implying a functional coupling between sodium channels (ENaC) and P2 receptors in the apical membrane of principal cells. Direct support for this proposition comes from the observation that ATPS reduces the open probability of apical sodium channels in the A6 cell line, which exhibits some properties of distal nephron cells (19). Other evidence from cell lines and nonrenal epithelia suggests that nucleotide-induced inhibition of ENaC is mediated, at least in part, by activation of Ca2+-dependent chloride channels (4, 14, 17), and in this context it is worth noting that luminal nucleotides can stimulate chloride secretion in cultured cell lines (4, 7). However, as pointed out elsewhere (17), chloride secretion is not a feature of native distal nephron epithelium. Nor, indeed, is nucleotide-induced inhibition of the amiloride-sensitive short-circuit current in mouse CCT dependent on an increase in intracellular calcium (16).
In view of uncertainties arising from the use of in vitro models, the present study was designed to address the question of whether luminal nucleotides could influence collecting duct sodium transport in the intact animal. For this we used ATPS, partly because it is only slowly hydrolyzed by native nucleotidases and partly because it is a broad-spectrum P2 receptor agonist, known to be effective (with varying degrees of potency) at P2Y1,2,4,and11 and P2X16 subtypes (10, 11). A concentration of 1 mM was employed, the rationale being that a maximal effect in vitro is achieved at 100 μM and cortical collecting ducts accept fluid from several (approximately 10) distal tubules.
In experiments in rats on a normal sodium intake, we could find little evidence for an effect of luminal ATPS on sodium reabsorption. It is, of course, theoretically possible that endogenous ATP, perhaps released in response to a change in tubular flow or shear force associated with the microperfusion procedure (17), was already exerting a major effect, but a more likely explanation for the negative finding is the sparseness of apical ENaC in the distal nephron of sodium-replete rats (20). We therefore repeated the procedure in animals that had been maintained on a low-sodium diet for 1 wk. As already indicated, this maneuver increased amiloride-sensitive sodium reabsorption in the collecting duct, consistent with increased expression of ENaC. In these animals, luminal ATPS elicited a significant reduction in collecting duct sodium reabsorption. The resistance of ATPS to hydrolysis argues against stimulation of P1 (adenosine) receptors; our results therefore provide the first in vivo demonstration of a functional role for apical P2 receptors in the collecting duct. That the effect was seen only in sodium-restricted animals conforms with the putative coupling between P2 receptors and ENaC. An alternative, hypothetical explanation for the response being confined to sodium-restricted rats is upregulation of P2 receptors in the distal nephron. However, a preliminary immunohistological investigation in our laboratory could find no obvious change in the pattern of distal nephron P2 receptor distribution in low-sodium rats (Turner CM, unpublished observations).
The change in urinary 22Na recovery in response to luminal ATPS, although consistent, was modest (increasing from 14 to 28%). It is possible that this figure underestimates the potential importance of luminal nucleotides in controlling collecting duct sodium reabsorption, because endogenous ATP release might already have been exerting an inhibitory effect on ENaC (although a baseline 22Na recovery of only 14% indicates that any such effect was minor). Whether endogenous nucleotide concentrations within the collecting duct ever achieve the values needed to exert a maximal inhibitory effect cannot be answered at present. Preliminary experiments in our laboratory (24) indicate that ATP concentrations in distal tubular fluid are very low (50 nM). However, assuming that ATP is released by epithelial cells, its concentration in the immediate vicinity of apical receptors may be considerably higher, as ectonucleotidases are present throughout the distal nephron (15, 29).
Accepting that P2 receptor stimulation by ATPS was able to inhibit ENaC-mediated sodium reabsorption in the collecting duct, the question arises: Which P2 receptor subtype(s) is/are responsible In the following discussion of collecting duct P2 receptor subtypes, we will limit the potential for confusion by confining our comments to native tissue; even then, some discrepancies exist. In mouse CCT, pharmacological profiling, based on a variety of functional responses to a range of applied nucleotides, points to the existence of apical P2Y2 and/or P2Y4 receptors (8, 16, 18). In rabbit CCT, the situation is less clear: one study (34) reported pharmacological evidence for apical P2Y2 or P2Y4 receptors, whereas another (8) could find no response to luminal ATP or UTP. The collecting duct of the rat has been more widely studied, using a variety of approaches. Pharmacological profiling suggests that P2Y1 and P2Y2 (and/or P2Y4) receptors are present in CCT and outer medullary collecting duct (OMCD) (6) and P2Y2 and/or P2Y4 receptors in inner medullary collecting duct (IMCD) (9). Messenger RNA for P2Y1, P2Y2, P2Y4, and P2Y6 receptors has been identified in OMCD (2, 3), while Kishore and colleagues (13) provided evidence not only for P2Y2 mRNA but also for P2Y2 receptor protein in IMCD. A recent immunohistological study of the distribution of P2 receptor proteins along the rat nephron has identified P2X5 receptors in MCD and low-level expression of P2X4 and P2X6 receptors throughout the collecting duct. The only P2Y receptor identified in the collecting duct was P2Y2, which, intriguingly, was confined to intercalated cells of the MCD (28).
As already indicated, ATPS can act on a wide range of P2 receptor subtypes. In an attempt to identify the subtype(s) involved in the inhibition of ENaC-mediated reabsorption, we employed a series of more selective agonists, although it is important to bear in mind that no agonist is absolutely selective for a given subtype and that current information about agonist selectivity is incomplete. On the basis of in vitro studies in mice, the obvious candidate receptor was P2Y2, which is pharmacologically similar to P2Y4 (5). We therefore used diadenosine tetraphosphate (Ap4A), whose P2Y agonism is limited to subtypes P2Y2 and (less potently) P2Y4, although it can also stimulate P2X15 receptors (10, 11); and the synthetic dinucleotide Cp4U, another potent and relatively stable agonist of P2Y2 receptors, again with a small effect on P2Y4 receptors (11, 35). The somewhat surprising finding was that neither of these compounds had any effect in our preparation, arguing strongly against P2Y2 or P2Y4 receptor mediation of the ATPS-induced inhibition of collecting duct sodium reabsorption in the rat. It is not easy to explain the discrepancy between our in vivo findings and previous in vitro observations. One possibility is a species difference in P2 receptor distribution and/or function, given that all the in vitro evidence implicating P2Y2 receptors has come from mouse models (7, 16, 26). As noted above, we have been unable to detect P2Y2 receptor protein in collecting duct principal cells of the rat (28). However, an obstacle to acceptance of this explanation is the earlier immunocytochemical study of Kishore et al. (13) in which P2Y2 receptor protein was reported in principal cells (as well as thin limbs of Henle and vascular structures) of tissue blocks prepared from inner medulla of rat kidney.
To assess the possible contribution of P2Y1 receptors in our preparation, we applied 2meSADP, commonly used as a selective and potent agonist of this receptor subtype (22), although it can also stimulate P2Y11 and P2Y12 receptors (11). However, this agonist was also without significant effect on 22Na recovery. Finally, the possible involvement of P2X receptors was tested by using BzATP, which acts on most P2X subtypes in addition to P2Y2 and P2Y11 receptors (11, 33). In this context, a recent in vitro study in our laboratory, in which ENaC and P2X receptors were coexpressed in Xenopus laevis oocytes, found that stimulation of P2X4 receptors, previously identified in rat collecting duct (28), could inhibit the amiloride-sensitive current characteristic of ENaC (31). Nevertheless, in our in vivo system, introduction of BzATP produced no consistent change in 22Na recovery.
Taken as a group, the four relatively selective agonists we used encompassed the entire range of documented activities of ATPS. On the face of it, therefore, none of the individual P2 receptor subtypes known to be present in the kidney appeared to mediate the inhibition of ENaC. A factor that should be considered here, however, is the tendency for P2X receptors to polymerize, either as homomeric assemblies of identical P2X subtypes or as heteromeric assemblies of more than one subtype. Of the individual P2X subtypes so far identified in rat collecting duct (28), the heteromeric assemblies P2X4/5, P2X4/6, and P2X5/6 can be formed (27), and it is even possible that all three subtypes might coassemble. The agonist profiles of such heteromers may differ from those of the individual constituent units, and we have recently found, using the X. laevis oocyte expression system, that ATPS (but none of the other agonists used in the present in vivo study) is equipotent with ATP in activating the P2X4/6 heteromer (32). Significantly, we had previously shown that activation of the P2X4/6 heteromer in this system caused downregulation of coexpressed ENaC (31). Thus, although still speculative at this stage, this line of reasoning might offer an explanation for the lack of success in identifying the receptor responsible for inhibition of ENaC in our in vivo study.
In conclusion, the present investigation in the intact rat has provided the first direct evidence for a functional role of apical P2 receptors in modulating collecting duct sodium reabsorption in vivo. In contrast to previous in vitro findings in the mouse, the inhibition of ENaC was not mediated by P2Y2 receptors; the identity of the receptor subtype(s) responsible remains to be determined.
GRANTS
We thank the Wellcome Trust and St. Peter's Trust for Kidney, Bladder and Prostate Research for financial support and Inspire Pharmaceuticals for the gift of Cp4U.
ACKNOWLEDGMENTS
The authors thank Drs. B. King and S. Wildman for helpful discussion.
FOOTNOTES
Address for correspondence: D. G. Shirley, Dept. of Physiology and Centre for Nephrology, Royal Free and Univ. College Medical School, Hampstead Campus, Rowland Hill St., London NW3 2PF, UK (E-mail: david.shirley@ucl.ac.uk)
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.
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