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BASIC RESEARCH |
* Department of Veterinary Basic Sciences, The Royal Veterinary College, and Department of Physiology and Centre for Nephrology, University College London, London, United Kingdom; and Department of Pharmacology, New York Medical College, Valhalla, New York, New York
Correspondence: Dr. Scott S.P. Wildman, Department of Veterinary Basic Sciences, Royal Veterinary College, Camden Campus, Royal College Street, London NW1 0TU, UK. Phone: +44-0-20-7121-1903; Fax: +44-0-20-7468-5204; E-mail: swildman{at}rvc.ac.uk
Received for publication April 13, 2007. Accepted for publication November 21, 2007.
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Abstract |
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Introduction |
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TopAbstractIntroductionRESULTSDISCUSSIONCONCISE METHODSDISCLOSURESREFERENCES |
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Activation of P2R can alter the activity of certain epithelial cell membrane transport proteins.1,2,6 A key transport protein shown to be affected by the activation of P2R is the amiloride-sensitive epithelial Na+ channel (ENaC).2,6–8 ENaC is expressed at the apical membrane of principal cells (PC) in the collecting duct (CD), and its activity is an important factor in the regulation of arterial BP, because it determines the extent of Na+ reabsorption from tubular fluid along the distal nephron.9,10
Given the importance of ENaC in controlling arterial BP, it is perhaps not surprising that there have been several studies on the modulation of its activity by P2R in the CD. To date, P2R-mediated modulation of ENaC activity has been demonstrated by means of electrophysiologic techniques in murine and amphibian immortalized renal CD/distal nephron cell lines7,8,11–15 and in the Xenopus oocyte heterologous expression system (using recombinant rat P2XR and ENaC proteins).16 It has also been reported in the CD of rat17 and mouse18,19 using in vivo and in vitro luminal perfusion studies, respectively. The prevailing view from these studies is that P2R inhibit ENaC activity; however, open issues remain with regard to the P2R subtype(s) involved, the membrane localization of P2R in the CD, and the intracellular mechanism(s) responsible for inhibition.
To address these issues, we investigated the relationship between the activation of apical P2R and ENaC activity in PC (in situ) in microdissected split-open rat CD, using whole-cell patch-clamp electrophysiology, in combination with real-time PCR and immunohistochemical techniques. Understanding the possible regulation of ENaC activity by P2R will extend our insight into the local and potentially "intracrine" control of renal tubular sodium transport and may also provide a novel therapeutic target for abnormal renal sodium transport in hypertension.
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RESULTS |
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TopAbstractIntroductionRESULTSDISCUSSIONCONCISE METHODSDISCLOSURESREFERENCES |
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Immunohistochemical Co-localization of P2R and ENaC Subunits
Positive immunofluorescence (localized to membrane domains and/or intracellular regions of PC and/or intercalated cells [IC]) was seen for P2X2, 4, 5, and 6 ion channel subunits and P2Y2, 4, 6, 11, 12, and 13 metabotropic receptors in the CD of Na+-replete rats (identified by positive aquaporin 2 [AQP2] staining). The same P2R subtypes were localized to the CD of Na+-restricted rats but with the addition of P2X1 and loss of P2X5 ion channel subunits (Table 1). For all P2R antibodies used, preincubation with their respective antigenic peptides abolished positive immunofluorescence (see Supplemental Data 1). Positive immunofluorescence was also seen for 
-, β-, and 
-ENaC subunits in the CD of both Na+-replete and Na+-restricted rats; this immunofluorescence appeared stronger in the PC of Na+-restricted rats.
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-ENaC. Strong basolateral immunostaining for P2X4 and 6 was seen in all PC; basolateral staining for P2Y2 was evident only in medullary CD PC. Punctate intracellular staining (i.e., staining that could not be confidently localized to the plasma membrane of PC) was seen for P2X2 and 5; P2Y2, 6, 12, and 13 (Table 1); and β- and -ENaC.
In PC from Na+-restricted rats, consistently positive β-ENaC and -ENaC staining became evident in the apical domain throughout the CD, and intracellular immunostaining for P2X5 disappeared. Localization and staining of all other P2X subunits was unchanged, except for the appearance of weak expression of P2X1 subunits in the inner medullary CD (Figure 1, A and B). With respect to P2YR, P2Y2, 4, 11, 12, and 13 staining was unchanged (Figure 1C) and P2Y6 showed restricted intracellular staining (see Table 1). As an incidental finding, positive P2X1 staining became evident in IC, but this was not investigated further.
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We failed to detect significant levels (i.e., >1 arbitrary unit; corresponding to <10 P2R transcripts per cell; for calculation, see Supplemental Data 2) of P2X2, 3, 5, and 7 and P2Y1 mRNA in rats maintained on either of the Na+ diets (n = 6), which broadly agrees with the immunohistochemical findings. In contrast, significant amounts of mRNA (all of a similar value [i.e., approximately 2 arbitrary units]; for calculation of number of transcripts per cell, see Supplemental Data 2) were detected for P2X4 and P2Y2 and 6 in CD of Na+-replete rats (Figure 2). CD from Na+-restricted rats showed a significant increase in abundance of P2X4 mRNA (by two-fold; n = 6; P < 0.01) but no change in P2Y2 or P2Y6 mRNA levels. Whereas P2X1 and 6 and P2Y4 mRNA levels were deemed insignificant in Na+-replete rats, Na+-restricted rats showed marked abundance of mRNA for each of these subtypes (Figure 2).
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Figure 3 shows ATP- and other P2R agonist-evoked (all at 10 µM) whole-cell inward currents in voltage-clamped (holding potential [Vh] = –60 mV) PC in situ from rats maintained on a Na+-replete diet. Agonists were selected according to the P2R identified in our immunohistochemical and real-time PCR studies (see Table 2).
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S; n = 5), 232 ± 45 pA (Ap6A; n = 5), 600 ± 123 pA (UTP; n = 8), 594 ± 21 pA (2meSATP; n = 5), 28 ± 11 (2meSADP; n = 5), and 114 ± 39 pA (BzATP; n = 3; Figure 4A, data normalized for cell size). Currents evoked by the application of Ap6A, UTP, BzATP, and 2meSADP were completely abolished after preincubation (3 min) and co-application of DIDS (4,4'-Di-isothiocyanatostilbene-2,2'-disulfonic acid; a Ca2+-activated Cl– channel blocker; 100 µM; n = 3 in each case; example in Figure 3B), suggesting that their action was mediated through G protein–coupled P2YR and the involvement of Ca2+-dependent chloride "reporter" currents. Currents evoked by 2meSATP were unaffected after DIDS treatment (Figure 3B).
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S; n = 5), 273 ± 48 pA (UTP; n = 6), 388 ± 21 pA (2meSATP; n = 5), and 224 ± 38 (BzATP; n = 3; Figure 4B). Ap6A and 2meSADP (both 10 µM) failed to evoke significant inward currents (i.e., currents >20 pA; n = 5). Currents evoked by the application of UTP and BzATP were abolished after preincubation (3 min) and co-application of DIDS (100 µM; n = 3 in each case; data not shown); currents evoked by 2meSATP were unaffected after DIDS treatment.
Concentration-response (C/R) curves were constructed for the range of nucleotide agonists used. As shown in Figure 5A, in Na+-replete rats, agonist C/R relationships demonstrated that ATPS, UTP, 2meSATP, and BzATP all are full agonists, with all but the last having similar potency to that of ATP. EC50 values were as follows: ATP 1.3 ± 0.8 µM (n = 5); ATPS 2.8 ± 1.1 µM (n = 5); UTP 2.8 ± 0.9 µM (n = 5); 2meSATP 1.3 ± 0.9 µM (n = 4); BzATP 18 ± 3 µM (n = 3). In contrast, Ap6A was only a partial agonist and 2meSADP was inactive at concentrations up to 1 mM.
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S 0.4 ± 0.3 µM (n = 5); UTP 0.9 ± 0.4 µM (n = 5); 2meSATP 0.4 ± 0.4 µM (n = 5); and BzATP 8 ± 2 µM (n = 3).
Current/Voltage Relationships
Steady-state current/voltage (I/V) relationships for whole-cell currents from PC were investigated for amiloride-sensitive currents (ENaC-mediated currents [Iam-s]). Figure 6 shows typical amiloride-sensitive I/V relationships recorded from Na+-replete and Na+-restricted rats. Data for I/V curves were calculated as the difference between total current amplitude and current amplitude in the presence of 10 µM amiloride (blocker of ENaC activity; as shown in Figure 6A from a Na+-restricted rat). I/V curves for Iam-s showed inward rectification, with mean reversal potentials (Erev) of –4 ± 4 mV (n = 6) and 58 ± 7 mV (n = 46) in PC from Na+-replete and Na+-restricted rats, respectively (Figure 6B). In Na+-replete rats, Iam-s was close to zero (n = 6), indicating a paucity of apical ENaC. The effect of amiloride (in Na+-restricted rats) was found to be reversible, but, with time, Iam-s decreased and often became undetectable after 20 min.
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S, or 2meSATP, Iam-s was found to be significantly reduced in amplitude (by 62 ± 6, 52 ± 6, 29 ± 7, and 31 ± 4%, respectively, on the basis of calculations using values at –60 mV; P < 0.01; n = 12 in all cases), yet the Erev value and inward rectification did not change significantly (Figure 7B). The inward currents evoked by Ap6A, 2meSADP, and BzATP failed to alter significantly the amplitude or Erev of subsequent amiloride-sensitive I/V relations (n = 12). Erev values for Iam-s were as follows: 58 ± 7 mV (control), 57 ± 6 mV (post-ATP), 56 ± 8 mV (post-UTP), 62 ± 4 mV (post-ATPS), 58 ± 9 mV (post-2meSATP), 57 ± 6 mV (Ap6A), 58 ± 5 mV (2meSADP), and 60 ± 12 mV (BzATP).
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S significantly increased the amplitude of amiloride-sensitive I/V relationships by 77 ± 4 and 64 ± 7% (P < 0.01) without altering inward rectification or mean reversal potential. Decreasing [Na+]ext had no significant effect on the UTP-mediated decreases in Iam-s amplitude but decreased the inhibitory effect of ATP from 62 ± 6% inhibition to 30 ± 9% inhibition (P < 0.05; n = 5), without altering the Erev or inward rectification. Erev values for Iam-s were as follows: 53 ± 6 mV (control), 56 ± 6 mV (post-ATP), 49 ± 10 mV (post-UTP), 59 ± 6 mV (post-ATPS), 58.5 ± 9 mV (post-2meSATP), 52 ± 5 mV (Ap6A), 53 ± 5 mV (2meSADP), and 52 ± 7 mV (BzATP).
When extracellular Na+ concentrations were changed from 145 to 50 mM, maximal amplitudes of DIDS-insensitive currents evoked by ATP, 2meSATP, and ATPS (100 µM, Vh = –60 mV) were significantly increased, by 11 ± 4, 32 ± 9, and 36 ± 5%, respectively (P < 0.05; n = 5). In contrast, the amplitude of UTP-evoked currents did not change significantly. The C/R curves for ATP, 2meSATP, and ATPS were not significantly shifted when [Na+]ext was decreased (data not shown).
Mechanisms of Iam-s Inhibition and Potentiation
Previous studies demonstrated that activation of phospholipase C β (PLCβ), which can be blocked by U73122, resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and/or activation of phosphatidylinositol-3 kinase (PI3K; which can be blocked by wortmannin or LY294002), may be involved in P2R-mediated inhibition of ENaC activity.14,16,25,26 With these observations in mind, the molecular mechanisms of P2R-mediated inhibition and potentiation of Iam-s amplitude were investigated pharmacologically in Na+-restricted rats. Two P2R agonists were selected for investigation, on the basis of their modulation of Iam-s: 2meSATP (predicted in this system to target apical P2X4 and/or 4/6 receptor) and UTP (predicted to target P2Y2 and/or 4 receptor).
When P2R were activated by UTP (10 µM) and [Na+]ext was 145 mM, preincubation (3 min) and co-application with U73122 (100 nM) led to a significant decrease in the percentage inhibition of Iam-s amplitude from 53 ± 8 to 30 ± 8% (Vh = –60 mV; P < 0.01; n = 4; Figure 8A), with no change in the amplitude of the UTP-evoked current. Preincubation/co-application with wortmannin (100 nM), U73343 (100 nM; the inactive form of U73122), or LY294002 (50 µM; data not shown) had no significant effect on the degree of UTP-mediated inhibition of Iam-s (n = 3 in all cases; Figure 8A). When P2R were activated by 2meSATP (10 µM), preincubation and co-application of wortmannin (100 nM), U73122 (100 nM), U73343 (100 nM), or LY294002 (50 µM; data not shown) did not significantly affect Iam-s inhibition (n = 3; Figure 8B).
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P2R Expression in the CD
We characterized pharmacologically and immunohistochemically P2R expressed in PC of the cortical and outer medullary CD, in both Na+-replete and Na+-restricted rats. We consistently demonstrated apical expression of P2X4 and 6 subunits and P2Y2, 4, 6, and 11 receptors in Na+-replete rats and apical expression of P2X4 and 6 subunits and P2Y2, 4, and 11 receptors in Na+-restricted rats in which functional ENaC was expressed (see Table 1). Furthermore, using real-time PCR, we demonstrated that dietary Na+ restriction increases mRNA levels of apically expressed P2X4 and 6 subunits and P2Y4 receptors but not of P2Y2 receptors (see Figure 2).
Our immunohistochemical findings demonstrating apical localization of P2R in rat PC are broadly in accordance with previous studies that collectively reported P2X4 and 6 and P2Y2, 4, 6, and 11 localization/expression in native epithelia of rats, mice, and rabbits and in epithelial cell lines.6,27–39 They also extend the findings of an earlier study in which, using Na+-replete rats and a limited number of commercially available P2R antibodies, we demonstrated a variety of P2R expressed throughout the nephron.40
P2 Receptor-Mediated Inhibition of Iam-s Amplitude
It has already been established that the activation of apically expressed P2R by extracellular nucleotides can decrease Iam-s amplitude in the CD,11–14,17–19 but argument remains over the P2R subtype(s) responsible. Previous pharmacologic profiling studies using mouse isolated CD reported that apical G protein–coupled P2Y2-like receptors mediated ATP-evoked inhibition of ENaC activity.18,19 Furthermore, distal nephron cell line studies using mouse M1 and Xenopus A6 cells suggested the exclusive involvement of an apical P2Y2-like subtype.11,12,14 In contrast, in a more recent in vivo microperfusion study in the rat, we proposed an apical ionotropic ATPS-sensitive P2XR-mediated effect,17 likely to be via a P2X4/6 receptor ion channel.41 In this context, we have demonstrated that certain recombinant rat P2XR (including P2X4 and 4/6) can inhibit ENaC activity in Xenopus oocytes.16 Finally, a study using mouse mIMCD-K2 cells suggested the involvement of both P2X (including P2X4) and P2Y (including P2Y2) subtypes.13
By using a variety of P2R agonists (and taking account of our immunohistochemistry and real-time PCR findings), this study provides evidence for both DIDS-insensitive P2XR-mediated (P2X4 and/or 4/6) and DIDS-sensitive P2YR-mediated (P2Y2 and/or 4) decreases in Iam-s amplitude in PC from rat CD. Thus, our findings are consistent with previous reports that suggested P2Y2 and/or ATPS-sensitive P2XR (including P2X4) involvement.11–14,17–19 In addition, we suggest that additional P2R subtypes/subunits are involved (i.e., rat P2Y4, which is pharmacologically similar to rat P2Y2,42 and P2X6). We have been unable to distinguish between P2X4 and P2X4/6 subtypes and between P2Y2 and P2Y4 receptors because of their similar pharmacologic profiles and the lack of any truly selective P2R agonists and antagonists3,5,42; however, our real-time PCR studies suggested a greater P2X4/6 and P2Y4 involvement, because mRNA levels for these subunits/receptors were significantly increased after dietary Na+ restriction and consequent ENaC upregulation (see Figure 2).
It is clear from our experiments that P2X-mediated inhibition (i.e., evoked by ATPS or 2meSATP) is approximately 50% less than that mediated by P2Y receptors (i.e., evoked by UTP; see Figure 7A), which may explain why several studies have failed to implicate clearly an inhibitory P2X effect (i.e., the P2Y effect may mask the P2X effect).
P2X Receptor–Mediated Potentiation of Iam-s Amplitude
The most striking finding of this study is that when [Na+]ext was reduced (from 145 to 50 mM; one where [Na+]ext-mediated inhibition of P2X4 and/or 4/6 activity is reduced),23,24 P2X4 and/or 4/6 activation did not inhibit ENaC activity but potentiated it. To our knowledge this is the first report of apical P2XR potentiating ENaC activity in the CD and of a P2R differentially regulating an ion channel. A possible reason for this is that previous electrophysiologic and perfusion studies investigating the effect of P2R activation on ENaC activity used extracellular solutions containing 145 mM Na+ (to optimize Na+ absorption).11–13,18,19 Our novel observation when using 50 mM [Na+]ext is important because this intraluminal Na+ concentration is much more physiologic (with respect to the distal nephron) than the previously used 145 mM.
The only other investigation to have used a physiologic Na+ concentration is a microperfusion study of the rat CD in vivo, in which a series of P2R agonists were tested intraluminally.17 In that study, a small but consistent inhibitory influence of ATPS on CD Na+ reabsorption was found. It is difficult to reconcile this in vivo observation with our finding in patch-clamped rat PC that activation of P2X4 and/or 4/6 receptors (by the application of ATPS) caused potentiation of ENaC activity at a luminal Na+ concentration of 50 mM. It is conceivable that subtle differences between the experimental conditions (e.g., in the in vivo study, P2R agonists were applied for approximately 5 min and at a dosage of 1 mM) might account for the discrepancy and that in vivo inhibitory effects of P2YR activation were able to override stimulatory effects of P2XR activation, which conceivably could have been desensitized by the high concentration of agonist, although it should be noted that Shirley et al.17 found that relatively selective agonists of P2Y2 and 4 receptors had no detectable effect on Na+ reabsorption.
Mechanism of P2R-Mediated Regulation of ENaC
As with the P2R subtype(s) responsible for inhibition of ENaC in the CD, there is also controversy over the proposed mechanism(s). Proposals have included activation of PLC14,25; decreased levels of PIP2 in the plasma membrane as a result of PLC- and/or protein kinase C–induced hydrolysis26; increases in intracellular Ca2+ after inositol IP3 and diacylglycerol formation26; activation of protein kinase C after IP3 and diacylglycerol formation26; influxes of extracellular Ca2+ ions16; increases in intracellular Na+ and downstream involvement of Go proteins16,43; activation of PI3K16; and changes in intracellular Cl– levels, which in some cases involved Gi proteins.11,16,25
In this study, we investigated the mechanism(s) responsible for both P2R-mediated inhibition and potentiation of ENaC in PC of the CD in situ. Our data suggest the involvement of PLC activation in the inhibition of Iam-s by P2YR activation, irrespective of [Na+]ext concentration (given that P2Y-mediated decreases in Iam-s amplitude can be inhibited by U73122), which agrees with mechanisms already proposed.14,25,26 It is interesting that P2Y-mediated inhibition was completely abolished by U73122 when [Na+]ext was low but only reduced when [Na+]ext was high, which suggests that more than one mechanism is responsible for P2Y-mediated inhibition of ENaC when [Na+]ext is high (but not involving PI3K activation, because wortmannin and LY294002 have no effect; see Figure 8A).
The results with wortmannin (and LY294002) suggest the involvement of PI3K activation in the potentiation of Iam-s by P2X4 and/or 4/6 activation when [Na+]ext was low (50 mM). In this respect, activation of PI3K by P2X4 receptor assemblies was previously described in embryonic stem cells and microglia.44,45 When [Na+]ext was high (145 mM), P2X4 and/or 4/6 activation resulted in an inhibition of Iam-s, yet this was not altered by U73122, wortmannin, or LY294002. In contrast, when P2X4 and/or 4/6 receptors were activated in the absence of extracellular Na+ (i.e., 2meSATP was prepared in a Na+-free Ringer solution), Iam-s was not inhibited (data not shown), indicating a requirement for Na+ influx in P2X-mediated inhibition of ENaC. Furthermore, we showed previously that activation of P2X4 and P2X4/6 receptors when [Na+]ext was relatively high (100 mM) inhibited ENaC activity through an increase in intracellular Na+ and Ca2+,16 which may also be the case here.
Is the P2X4 and/or 4/6 Receptor a Na+ Sensor?
The results of this study indicate that whereas apical P2Y2 and/or 4 receptors are consistently inhibitory with respect to ENaC activity, P2X4 and/or 4/6 receptors have the ability to inhibit or potentiate ENaC, depending on the prevailing luminal Na+ concentration. Under normal circumstances, where functional ENaC is sparse, the luminal Na+ concentration is approximately 50 mM throughout the distal tubule46 but may rise to approximately 100 mM along the CD. In conditions of Na+ restriction, although the Na+ concentration in the distal tubule is little affected, it falls progressively in the CD and may be as low as 3 mM in the final urine.47 Under the latter condition, it would be expected that stimulation of apical P2X4 and/or 4/6 receptors should increase apical ENaC activity and facilitate Na+ reabsorption in the CD (see Figure 9).
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It is not possible to say at present whether this P2R-mediated modulation of ENaC activity occurs by altering the channel's apical membrane insertion or retrieval (as seen in the Xenopus oocyte expression system with regard to ENaC inhibition16) or by changing its open probability. What is clear is that through its action on functional ENaC activity, the apical P2R system has the potential to alter Na+ reabsorption significantly and in so doing may affect sodium balance and arterial BP. That hypertension is a feature of both P2X4 and P2Y2 knockout mice suggests an important interplay within the P2R regulatory system and sodium homeostasis in vivo.50,51
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CONCISE METHODS |
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Antibodies
Polyclonal primary antibodies (Ab) recognizing intracellular domains of rat P2X subunit proteins were obtained from Prof. G. Burnstock (Autonomic Neuroscience Institute, UCL, London, UK; for epitope sequences, see Turner et al.40); P2Y1, 2, 4, 6, 11, 12, 13, and 14 receptor Ab were purchased from Alomone Laboratories (Jerusalem, Israel); , β-, and -ENaC Ab were obtained from Prof. B. Rossier (University of Lausanne, Lausanne, Switzerland; for epitope sequences, see Lin et al.54); and AQP2 Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Noncommercially available P2X Ab were previously demonstrated to show subunit specificity.55
Immunohistochemistry
Rats were terminally anesthetized by intraperitoneal injection of sodium pentobarbitone ([200 mg/ml] used at 60 mg/kg), and the kidneys were perfusion-fixed with paraformaldehyde (4%). Kidneys were removed, embedded in OCT compound (Agar Scientific, Stansted, UK), and snap-frozen. Tissues were sectioned at 8 µm using a cryostat (Reichert Jung CM 1800, Milton Keynes, UK). The slides were stored at –70°C and thawed at room temperature for 10 min before treatment.
Slide-mounted sections were washed in PBS, treated with 10% donkey serum PBS (1 h), incubated overnight at 4°C with either P2R or ENaC primary Ab, and then incubated for 2 h with AQP2 primary Ab. Binding was revealed with FITC-conjugated and Cy3-conjugated (Jackson ImmunoResearch Laboratories, West Grove, PA) secondary Ab. Positive AQP2 immunostaining was used to identify PC of the CD.
Slides were mounted using Citifluor mountant (Agar Scientific, Essex, UK) and examined using a Bio-Rad Radiance 2100 confocal laser scanning microscope linked to a Nikon E800 fluorescence microscope and using a x40, 1.3 NA, oil immersion objective. Controls for nonspecific binding of primary and secondary Ab were performed by preincubating immune sera with their respective immunogenic peptide (overnight) or by omitting the primary Ab for histochemical procedures, respectively.
Isolation of Tubules
Rats were terminally anesthetized by intraperitoneal injection of sodium pentobarbitone ([200 mg/ml] used at 60 mg/kg). The kidneys were removed, decapsulated, sliced into thin corticomedullary pyramids, and placed in an ice-cold dissection solution containing (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 2 glucose, and 10 Hepes (pH 7.4, NaOH). CD from the cortical and outer medullary regions were isolated under a dissecting microscope, as described previously.56 Where necessary, collagenase (NB4G; 1 mg/ml for 20 min; SERVA, Heidelberg, Germany) was used on pyramids to aid tubule isolation. CD were identified by their anatomy and morphology, as described previously.57
Real-Time PCR
RNA was extracted from pools of microdissected CD (total length 15 to 20 mm) from the cortical and outer medullary regions using an adapted guanidium thiocyanate-phenol-chloroform method.58 One microgram of total RNA was reverse-transcribed with 0.5 µg oligo(-dt) 12 to 18 primer and a first-strand cDNA synthesis kit (Superscript II RNase H– reverse transcriptase; Life Technologies BRL, Paisley, UK). The resulting cDNA transcripts were used for PCR amplification using a Roche Lightcycler (Roche Diagnostics, Mannheim, Germany) and QuantiTect SYBR Green PCR kit (Qiagen, West Sussex UK). Gene-specific primers for P2R and the constitutively expressed gene HPRT were used (Table 3).
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Flame Photometry
Upon removal of the kidneys, urine was collected from the bladder and the Na+ content was measured by flame photometry (model 543; Instrumentation Laboratory, Warrington, UK) to confirm that reduced dietary Na+ intake decreased urinary Na+ content.
Patch Clamp
Isolated tubules were immobilized on glass coverslips coated with poly-L-lysine (50 µg/ml in dissection solution) and transferred to a chamber (volume approximately 600 µl) mounted on the stage of an inverted microscope (Nikon Eclipse TE300). CD were opened with a sharpened micropipette to access the apical membrane. PC were identified by their flatness, abundance, and polygonal shape.
The whole-cell configuration of the patch-clamp technique was used; this was achieved through a nystatin perforated-patch method. For establishment of the perforated-patch, nystatin (50 to 100 µg/ml) was added to the pipette solution containing (in mM) 50 KCl, 90 K-gluconate, 3 MgCl2, 3 EGTA, and 10 Hepes (pH 7.2, KOH). The development of electrical access to the cell interior was monitored by following the nystatin-induced fall in access resistance (Ra). Experiments were initiated once Ra had fallen to a stable value of <35 M
(this typically took approximately 10 min) and subsequent series resistance compensation was applied. An Axopatch 200B patch-clamp amplifier (Axon Instruments, Sunnyvale, CA) and computer complete with pCLAMP 8.0 software (Axon Instruments) were used to store and analyze whole-cell currents. Patch pipettes were pulled from borosilicate glass capillaries (Harvard Apparatus, Edenbridge, UK) and had a resistance ranging between 4.0 and 6.0 M. The reference electrode was an Ag/AgCl pellet. Current signals were filtered at 5 kHz and displayed on an oscilloscope (20 MHz digital storage; Gould, Eichstetten, Germany). Liquid junction potentials were calculated using pCLAMP software, and appropriate corrections were applied.
Split-open CD were slowly superfused (4 ml/min, low enough to avoid mechanical disturbances and associated endogenous ATP release; 21 to 23°C) with bathing solution that contained (in mM) 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, and 10 Hepes (pH 7.4, NaOH) by a gravity-fed, continuous-flow system that allowed drug addition and washout. In experiments to assess the effect of altering [Na+]ext, Ringer solution was altered to contain (in mM) 50 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 Hepes, 90 TEA-Cl, ad 90 NMDG+ (pH 7.4, NaOH). P2R agonist-activated membrane currents were recorded at a Vh of –60 mV, sufficient to drive P2X-mediated inward cationic currents and P2Y-mediated Ca2+-dependent chloride currents. P2R agonists were applied for 30 s or until the current reached a peak, whichever was the longer, then washed out for 3 min to avoid rundown. Only cells that demonstrated an inward current amplitude (evoked by 10 µM ATP) >100 pA were used. Amiloride-sensitive whole-cell currents (Iam-s) were calculated as the difference between total currents and currents obtained in the presence of amiloride (10 µM). Steady-state I/V relations were determined for each whole-cell clamp by stepping the command voltage down to –60 mV and up to 80 mV from 0 mV in 20-mV increments for 100 ms.
Statistical Analyses
All data are presented as mean ± SEM; significance was evaluated by t test, paired or unpaired as appropriate (Instat 3.0; GraphPad Software, San Diego, CA), with P < 0.05 considered significant.
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Acknowledgments |
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Part of this work was presented at the American Society of Nephrology Renal Week; November 11 through 14, 2006; San Diego, CA.38
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Footnotes |
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Supplemental information for this article is available online at http://www.jasn.org/.
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S. S. P. Wildman, M. Boone, C. M. Peppiatt-Wildman, A. Contreras-Sanz, B. F. King, D. G. Shirley, P. M. T. Deen, and R. J. Unwin Nucleotides Downregulate Aquaporin 2 via Activation of Apical P2 Receptors J. Am. Soc. Nephrol., July 1, 2009; 20(7): 1480 - 1490. [Abstract] [Full Text] [PDF]
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Y. Zhang, R. D. Nelson, N. G. Carlson, C. D. Kamerath, D. E. Kohan, and B. K. Kishore Potential role of purinergic signaling in lithium-induced nephrogenic diabetes insipidus Am J Physiol Renal Physiol, May 1, 2009; 296(5): F1194 - F1201. [Abstract] [Full Text] [PDF]
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 O. Pochynyuk, V. Bugaj, T. Rieg, P. A. Insel, E. Mironova, V. Vallon, and J. D. Stockand Paracrine Regulation of the Epithelial Na+ Channel in the Mammalian Collecting Duct by Purinergic P2Y2 Receptor Tone J. Biol. Chem., December 26, 2008; 283(52): 36599 - 36607. [Abstract] [Full Text] [PDF]
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