 in these animals performed previously33,34 provide evidence that Cx30−/− in the distal nephron, particularly in intercalated cells, is important for ATP release into the urine. Our finding that the bulk of salt-sensitive ATP release is lost in Cx30−/− mice suggests, in addition, that this route of release may be particularly important during responses to changes in Na+ balance. This idea is consistent with the findings that renal Na+ excretion and pressure natriuresis are impaired in Cx30−/− mice and that these mice develop ENaC-dependent, salt-sensitive elevations in BP.34 Principal cells in the ASDN are another possible source of ATP release with release from these cells seeming to depend on the presence of the apical primary cilia.37,40 Interestingly, this monocilia may be involved in sensing urine flow, which rises upon increases in systemic Na+ levels. Other possible sources of urinary ATP potentially capable of affecting transport proteins in the distal nephron are upstream nephron segments, including the thick ascending limb.38,39 For instance, ATP released from the thick ascending limb in response to flow acts in a paracrine manner to mobilize Ca2+ in tubules from WT but not P2Y2−/− mice.39
Although the precise mechanisms that trigger ATP release into the urine in response to elevations in Na+ intake and the intrarenal source of this paracrine-acting ATP remain to be definitively established, the observation that increases in Na+ balance increase urinary [ATP] is consistent with previous findings.11,30,31 Micropuncture studies have shown that ATP levels in the ultrafiltrate of the proximal tubule climb as high as 300 nM with distal tubule levels being approximately 3.5-fold lower.41 In agreement with this, we found urinary ATP levels in the nanomolar range. As a result of the actions of ecto-ATPase and ecto-5′-nucleotidase and other extracellular enzymes capable of metabolizing ATP, quantification of [ATP] from urine in the bladder may underestimate, somewhat, actual values. Alternatively, water reabsorption distal to the site of ATP release may concentrate yielding ATP values modestly higher in the final urine compared with urine in the ASDN. Nevertheless, [ATP] concentrations consistent with those measured in urine have a marked effect on ENaC activity in its native environment in isolation from possible secondary effects, such as changes in perfusion pressure, urine flow, and other factors that may complicate interpretation. Thus, the [ATP] associated with high Na+ levels causes marked inhibition of ENaC, whereas the channel is uninhibited with [ATP] at lower levels of dietary Na+, results consistent with the regulation of ENaC by ATP signaling in the ASDN playing an important role in the renal Na+ excretory response to changes in systemic Na+ levels. Our findings showing that genetic deletion of the P2Y2 receptor, which is the primary target for paracrine ATP regulation of ENaC in the ASDN,11,28 impairs excretion of Na+ particularly during elevations of Na+ intake are in agreement with this idea. Our findings also agree with previous results showing that renal Na+ excretion is impaired in P2Y2−/− mice.29
Together, our results demonstrate that increasing systemic Na+ increases urinary [ATP] to levels capable of suppressing ENaC activity and facilitating Na+ excretion. RAAS has a similar role in mediating an ENaC response to salt: Increases in total body Na+, sensed as pressure and/or volume changes, suppress plasma renin activity and lower aldosterone levels, leading to less active ENaC and thereby facilitating Na+ excretion. ENaC activity thus changes inversely with respect to changes in systemic Na+ levels.
P2Y2−/− mice have facilitated renal Na+ reabsorption associated with suppressed plasma renin and aldosterone levels and lower-than-normal plasma K+.29 The latter is because K+ is secreted in the distal nephron to maintain charge neutrality as Na+ is reabsorbed via ENaC. This profile along with salt-sensitive changes in BP is the clinical manifestation of Liddle's syndrome: Gain of ENaC function.3,5,7,17 BP in P2Y2−/− animals, in the absence of any other perturbation, however, is salt resistant.29 This suggests little involvement of ENaC; however, on the basis of our previous work11,28 and results of this study, we conclude that regulation of ENaC by ATP functions in parallel and complements regulation by aldosterone. This means that ENaC activity can change, to some degree, in response to changes in Na+ intake when one but not both salt-sensing control systems is lost. In other words, ENaC activity, as shown in Figure 4, retains its ability to respond to changes in Na+ intake in the absence of mineralocorticoid or purinergic regulation but not when both are absent. Likewise, renal Na+ handling and BP are mostly normal in the absence of either but not of both salt-sensing regulatory mechanisms that govern ENaC activity. Consistent with this idea are our findings showing that relative Na+ excretion, although modestly lower in P2Y2−/− mice compared with WT mice, retains most of its dependence on Na+ balance and that Na+ excretion remains elevated during aldosterone escape despite elevated mineralocorticoid levels. In contrast, simultaneously disrupting regulation by both purinergic and mineralocorticoid signaling uncouples ENaC from being able to respond appropriately to Na+, giving it a high salt resistance and driving Na+ excretion to inappropriately low levels in the presence of high salt intake (see Figure 5).
The impaired ability of ENaC to respond to changes in Na+ intake leads to the salt-sensitive hypertension of P2Y2−/− mice treated with DOCA. As a result of aldosterone escape, WT mice treated with DOCA have BP that is not salt sensitive. We conclude that inhibition of ENaC by local purinergic signaling provides a mechanism for escaping the actions of aldosterone.
CONCISE METHODS
All chemicals and materials were from Sigma (St. Louis, San Antonio. For experiments, WT (C57BL/6), P2Y2−/− (backcrossed and inbred into the C57BL/6 background; described in detail previously11,28,29) and Cx30−/− (also inbred on the C57BL/6 background and described previously33,34) mice were maintained on a nominally Na+-free diet (<0.01% Na+; Harlan TEKLAD TD.90228), regular-Na+ diet containing 0.32% Na+ (Harlan TEKLAD TD.7912), or a high-Na+ diet (2% Na+; Harlan TEKLAD TD.92034) 1 week before experimentation. For some experiments, mice were administered a subcutaneous injection of 2.4 mg of DOCA dissolved in 150 μl of olive oil for 3 consecutive days before being killed.
Isolation of the ASDN containing CNT and CD suitable for electrophysiology has been described previously.10,11,28 Briefly, mice were killed by CO2 administration followed by cervical dislocation. Kidneys were immediately removed and were cut into thin slices (<1 mm), which were placed into ice-cold physiologic saline solution buffered with HEPES (pH 7.4). The ASDN was identified as merging of CNT into CD and was mechanically isolated from cortical sections of kidney slices by microdissection using watchmaker forceps under a stereomicroscope. Isolated ASDN was allowed to settle onto 5 × 5-mm coverglass coated with poly-l-lysine. Coverglass containing ASDN was placed in a perfusion chamber mounted on an inverted Nikon Eclipse TE2000 microscope and superfused with room temperature HEPES-buffered (pH 7.4) saline solution. ASDN were split open with two sharpened micropipettes controlled with different micromanipulators to gain access to the apical membrane and were used within 1 to 2 hours of isolation.
ENaC activity in principal cells of murine ASDN was determined in cell-attached patches on the apical membrane made under voltage-clamp conditions (−Vp = −60 mV) using standard procedures.10,28 Current recordings were made in a still bath with experimental reagents added directly to the recording chamber. Recording pipettes had resistances of 10 to 15 MΩ. Typical bath and pipette solutions were as follows: 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 5 mM glucose, and 10 mM HEPES (pH 7.4) and 140 mM LiCl, 2 mM MgCl2, and 10 mM HEPES (pH 7.4), respectively. For each experimental condition, ASDN from at least three mice were assayed. Gap-free single-channel current data from gigaohm seals were acquired (and subsequently analyzed) with an Axopatch 200B (Axon Instruments) or EPC-9 (HEKA Instruments) patch-clamp amplifier interfaced via a Digidata 1322A (Axon Instruments) to a PC running pClamp 9.2 software (Axon Instruments). Currents were low-pass-filtered at 100 Hz with an eight-pole Bessel filter (Warner Instruments). Unitary current (i) was determined, as normal, from all-point amplitude histograms fitted with single- or multi-Gaussian curves using the standard 50% threshold criterion to differentiate between events. Events were inspected visually before acceptance. Channel activity, defined as NPo, was calculated using the equation NPo = (t1 + 2t2 + … + ntn), where N and Po are the number of ENaC in a patch and the mean open probability of these channels, respectively, and tn is the fractional open time spent at each of the