Bradykinin acutely inhibits activity of the epithelial Na⁺ channel in mammalian aldosterone-sensitive distal nephron

Abstract

Activation of the renal kallikrein-kinin system results in natriuresis and diuresis, suggesting its possible role in renal tubular sodium transport regulation. Here, we used patch-clamp electrophysiology to directly assess the effects of bradykinin (BK) on the epithelial Na⁺ channel (ENaC) activity in freshly isolated split-opened murine aldosterone-sensitive distal nephrons (ASDNs). BK acutely inhibits ENaC activity by reducing channel open probability (P_o) in a dose-dependent and reversible manner. Inhibition of B2 receptors with icatibant (HOE-140) abolished BK actions on ENaC. In contrast, activation of B1 receptors with the selective agonist Lys-des-Arg⁹-BK failed to reproduce BK actions on ENaC. This is consistent with B2 receptors playing a critical role in mediating BK signaling to ENaC. BK has little effect on ENaC P_o when G_q/11 was inhibited with Gp antagonist 2A. Moreover, inhibition of phospholipase C (PLC) with U73122, but not saturation of cellular cAMP levels with the membrane-permeable nonhydrolysable cAMP analog 8-cpt-cAMP, prevents BK actions on ENaC activity. This argues that BK stimulates B2 receptors with subsequent activation of G_q/11-PLC signaling cascade to acutely inhibit ENaC activity. Activation of BK signaling acutely depletes apical PI(4,5)P₂ levels. However, inhibition of Ca²⁺ pump SERCA of the endoplasmic reticulum with thapsigargin does not prevent BK signaling to ENaC. Furthermore, caffeine, while producing a similar rise in [Ca²⁺]_i as in response to BK stimulation, fails to recapitulate BK actions on ENaC. Therefore, we concluded that BK acutely inhibits ENaC P_o in mammalian ASDN via stimulation of B2 receptors and following depletion of PI(4,5)P₂, but not increases in [Ca²⁺]_i.

precise matching between sodium intake and sodium excretion is necessary for normal blood pressure control. Positive salt balance results in expansion of the circulating volume and hypertension, whereas salt wasting leads to hyponatremia and volume contraction (23, 38, 54). The renin-angiotensin-aldosterone system (RAAS) controls sodium balance, in part, by fine-tuning sodium reabsorption at the aldosterone-sensitive distal nephron (ASDN) (12, 72). This segment includes the connecting tubule (CNT) and the cortical collecting duct (CCD). The epithelial Na⁺ channel (ENaC), which is localized to the apical plasma membrane of principal cells in the ASDN, is a critical end-effector of this regulation (11, 16, 23, 55). ENaC is uniquely positioned to respond to changes in systemic Na⁺ balance (23, 55). ENaC activity accounts for the majority, if not all, of the electrogenic Na⁺ reabsorption in the distal nephron and is in direct control by mineralocorticoid hormone aldosterone. Indeed, a clear link exists between ENaC dysfunction and blood pressure abnormalities in humans. Gain-of-function mutations of ENaC are associated with excessive salt retention and hypertension (Liddle syndrome), whereas loss-of-function mutations cause life-threatening salt wasting (particularly during childhood) and hypotension as exemplified by pseudohypoaldosteronism type 1 (21, 22, 26, 52–54, 60).

Kinins are local peptide hormones that mediate important biological processes, including pain sensation, edema formation, cell proliferation, hypotension, and natriuresis (5). Kallikrein is a serine protease that cleaves locally produced kininogen to generate kinins, including bradykinin (BK) (31, 39). The kallikrein-kinin system (KKS) is an important vasoregulatory component of cardiovascular and renal homeostasis (39, 70). Activation of KKS is thought to antagonize the effects of RAAS on blood pressure and sodium handling (59). The balance between these two systems affects salt sensitivity, circulating volume and vascular reactivity (56, 69). KKS and RAAS are functionally coupled at the level of angiotensin-converting enzyme (ACE). It is well-recognized that ACE inhibition is one of the most efficient pharmacological strategies for managing hypertension, congestive cardiac failure, and diabetic nephropathy (17, 25, 69). However, some of the beneficial actions of ACE inhibition cannot be attributed solely to the blocking of ANG II production. ACE is also a potent kininase that rapidly cleaves kinins to inactive peptides (59, 69). Therefore, activation of KKS may further contribute to the lowering of blood pressure during ACE inhibition. Consistent with this, mice lacking ACE activity exhibit hypotension and increased BK production (10).

In the kidney, KKS may also regulate water and electrolyte excretion, thus, contributing to control of plasma volume (37, 58). All components of the renal KKS are present in the distal part of the renal nephron. Specifically, kallikrein is actively synthesized in the CNT, whereas kininogen and B2 receptors are expressed mainly in the collecting duct (59). Stimulation of the renal KKS results in diuresis and natriuresis (49). Mice lacking receptors for BK develop salt-sensitive hypertension (1, 2). Circumstantial evidence supports the idea that Na⁺ reabsorption in the ASDN might be controlled by local kinins (37, 63, 65, 66). Lower urinary kallikrein levels have been detected in humans with essential hypertension and in strains of hypertensive rats (34–36, 46). Conversely, hypertensive mice and rats chronically or transiently overexpressing the human TK gene, which encodes kallikrein, present with hypotension (61, 73). Therefore, the renal KKS may play an important role in the long-term regulation of blood pressure under conditions of hypertensive insult, especially high-salt intake (1, 32, 68). It remains obscure, though, whether activation of the renal KKS directly inhibits ENaC activity to promote natriuresis.

BK interacts with G protein-coupled B1 and B2 receptors (19, 20). The biological effects of BK are mediated mainly through the B2 receptors that are constitutively expressed in smooth muscles, neurons, vascular endothelium, and kidney epithelial cells (29). In contrast, expression of B1 receptors is low during normal physiology but markedly upregulated by tissue injury and inflammation (33). B2 receptors are coupled mainly to Gα_q/11 and Gα_i. In endothelial cells, BK stimulates endothelial nitric oxide synthase (eNOS) via Ca²⁺-dependent mechanisms, leading to increased cGMP and NO production and vasodilation (30). This opposes the vasoconstricting actions of ANG II. In contrast, little is known about signaling mechanisms mediating BK actions on tubular sodium and water transport in the kidney.

In this study, we used patch-clamp electrophysiology in freshly isolated split-opened murine ASDNs to test whether ENaC is a direct target for BK signaling. We found that BK inhibits ENaC activity and open probability (P_o) in a reversible manner via activation of B2 receptors. Stimulation of the G_q/11-phospholipase C (PLC) cascade with subsequent PI(4,5)P₂ hydrolysis, rather than elevations in intracellular [Ca²⁺], decreases ENaC activity in response to BK signaling. Overall, our results provide the first direct support to the idea that increased BK production in response to KKS activation acutely inhibits ENaC activity to adjust Na⁺ reabsorption in the ASDN.

MATERIALS AND METHODS

Materials and animals.

All chemicals and materials were from Sigma (St. Louis, MO) unless noted otherwise and were of reagent grade. Animal use and welfare adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals following a protocol reviewed and approved by the Institutional Laboratory Animal Care and Use Committee of the University of Texas Health Science Center Houston. For experiments, male C57BL/6 mice (purchased from Charles River Laboratories, Wilmington, MA), 6–8 wk old, were used. Animals were maintained on a nominally Na⁺-free diet (<0.01% Na⁺; Harlan TEKLAD TD.90228) for at least 1 wk before experimentation.

Tissue isolation and culturing.

The procedure for isolation of the ASDNs containing the CNT and the CCD suitable for electrophysiology has been described previously (8, 41, 43). Briefly, mice were killed by CO₂ administration followed by cervical dislocation and the kidneys were immediately removed. Kidneys were cut into thin slices (<1 mm) with slices placed into ice-cold physiologic saline solution buffered with HEPES (pH 7.4). The ASDN was identified as merging of CNT into CCD and was mechanically isolated from cortical sections of kidney slices by microdissection using watchmaker forceps under a stereomicroscope. Isolated ASDN was attached to 5 × 5-mm cover glass coated with poly-l-lysine. A cover glass containing ASDN was placed in a perfusion chamber mounted on an inverted Nikon Eclipse Ti microscope and perfused with room temperature HEPES-buffered (pH 7.4) saline solution. ASDNs were split-open with two sharpened micropipettes, controlled with different micromanipulators, to gain access to the apical membrane. The tubules were used within 1–2 h of isolation.

The immortalized mouse collecting duct principal cell line mpkCCD_c14 has been described before (42). These cells, which contain functional mineralocorticoid receptors and ENaC, readily polarize, forming monolayers with high trans-epithelial resistance and avid aldosterone-sensitive Na⁺ reabsorption. In brief, these cells were cultured in defined medium on permeable supports (Costar Transwells, 0.4-μm pore, 24-mm diameter) as described previously (42). Cells were maintained in culture with fetal bovine serum and corticosteroids.

Electrophysiology.

ENaC activity in principal cells was determined in cell-attached patches on the apical membrane made under voltage-clamp conditions (−V_p = −60 mV) using standard procedures (41–43). Current recordings were made in a still bath with experimental reagents added directly to the recording chamber except for caffeine, which was added via perfusion. Drug application times are shown with bars on the top of representative single-channel traces. Recording pipettes had resistances of 8–12 MΩ. Typical bath and pipette solutions were (in mM) 150 NaCl, 5 mM KCl, 1 CaCl₂, 2 MgCl₂, 5 glucose, and 10 HEPES (pH 7.4) and 140 LiCl, 2 MgCl₂, and 10 HEPES (pH 7.4), respectively. For each experimental condition, ASDNs from at least three different mice were assayed. Gap-free single-channel current data from gigaohm seals were acquired (and subsequently analyzed) with an Axopatch 200B (Axon Instr.) patch-clamp amplifier interfaced via a Digidata 1440 (Axon Instr.) to a PC running the pClamp 10.2 suite of software (Axon Instr.). Currents were low-pass filtered at 100 Hz with an eight-pole Bessel filter (Warner Instr.). 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. ENaC activity was analyzed over a span of 40–120 s for each experimental condition after reaching a new steady state in response to a treatment. Channel activity, defined as NP_o, was calculated using the following equation: NP_o = (t₁ + 2t₂ +…+nt_n), where N and P_o are the number of ENaC in a patch and the mean P_o of these channels, respectively, and t_n is the fractional open time spent at each of the observed current levels. P_o was calculated by dividing NP_o by the number of active channels within a patch as defined by all-point amplitude histograms. For calculating P_o in paired experiments, N was fixed as the greatest number of active channels observed in control or experimental conditions. It is very unlikely that N can change in cell-attached experiments, so any detected effect must be an effect on P_o. The error associated with calculating P_o increases as this variable moves away from 0.5 and approaches 0 or unity. To ensure reliable calculation of P_o, we measured P_o in patches containing five or fewer channels.

Total internal reflection fluorescence microscopy.

The PI(4,5)P₂ reporter, GFP-PLC-δ-PH, is a chimera consisting of the PI(4,5)P₂-binding pleckstrin homology (PH) domain from PLC-δ1 conjugated to green fluorescent protein (GFP). The cDNA encoding this reporter was described earlier (42, 44). The PI(4,5)P₂ reporter was introduced into mpkCCD_c14 principal cells within a confluent monolayer with a biolistic particle delivery system (Biolistic PDS-1000/He Particle Delivery System; Bio-Rad). Use of this system has been described previously (42, 44, 64). In brief, mpkCCD_c14 cells were grown to confluence on permeable supports. After high-resistance monolayers that avidly transport Na⁺ were formed, cells were washed twice with physiologic saline, aspirated, and quickly bombarded (at the apical membrane) under vacuum with microcarriers coated with reporter cDNA. Medium was immediately returned to the cells, which were then placed within a tissue culture incubator for 2–3 days to allow expression of the PI(4,5)P₂ reporter. Bombardment had little disruptive effect on cellular and monolayer integrity, as determined by maintenance of Na⁺ transport and a high trans-epithelial resistance.

Fluorescence emissions from the PI(4,5)P₂ reporter at the apical membrane of mpkCCD_c14 cells within a confluent monolayer were collected using total internal reflection fluorescent (TIRF) microscopy (3, 4). Upon expression of the PI(4,5)P₂ reporter, 5 × 5-mm sections of the support were excised, inverted, and placed onto cover glass coated with poly-l-lysine. All TIRF experiments were performed in the TIRF microscopy core facility housed within the Department of Physiology at the University of Texas Health Science Center, San Antonio. We previously described imaging the GFP-PLC-δ-PH reporter using this core facility (41, 44, 64). In brief, fluorescence emissions were collected using an inverted TE2000 microscope with through-the-lens (prismless) TIRF imaging (Nikon). Samples were viewed through a plain Apo TIRF ×60 oil-immersion, high-resolution (1.45 NA) objective. Fluorescence emissions were collected through a 535 ± 25-nm bandpass filter (Chroma Technology) by exciting GFP with an argon-ion laser with an acoustic optic tunable filter (Prairie Technology) used to restrict excitation wavelength to 488 nm. Fluorescence images were collected and processed with a 16-bit, cooled charge-coupled device camera (Cascade 512F, Roper Scientific) interfaced to a PC running Metamorph software. This camera uses a front-illuminated EMCCD with on-chip multiplication gain. Images were collected once a minute with a 200-ms exposure time. Images were not binned or filtered with pixel size corresponding to a square of 122 × 122 nm.

[Ca²⁺]_i measurements.

Intracellular calcium levels were measured in cells of the split-open ASDNs using Fura-2 fluorescence ratiometric imaging as described previously (15, 75). Split-open ASDNs were loaded with Fura-2 by incubation with 2 μM Fura-2/AM in bath solution for 40 min at room temperature. Subsequently, the ASDNs were washed and incubated for an additional 10–15 min before experimentation. The ASDNs were then placed in an open-top imaging study chamber (Warner RC-10) with a bottom coverslip viewing window and the chamber attached to the microscope stage of an InCa Imaging Workstation (Intracellular Imaging). Cells were imaged with a ×20 Nikon Super Fluor objective and regions of interest were drawn for multiple cells. The Fura-2 fluorescence intensity ratio was determined by excitation at 340 and 380 nm and calculating the ratio of the emission intensities at 511 nm in the usual manner every 3 s. The changes in the ratio are reported as an index of changes in intracellular calcium (18).

Data analysis.

All summarized data are reported as means ± SE. Data from before and after treatment within the same experiment were compared with the paired t-test. Data from different experiments were compared with a Student's (2-tailed) t-test or a one-way ANOVA as appropriate. P ≤ 0.05 was considered significant. For presentation, current data from some cell-attached patches were subsequently software filtered at 50 Hz and slow baseline drifts were corrected.

RESULTS

We used patch-clamp electrophysiology in cell-attached configuration to test whether BK directly modulates ENaC activity in ASDNs. Figure 1A documents a representative current trace containing a single ENaC before, during application of BK (500 nM), and following washout with regular bath solution. As is clear from the representative paired experiment and the summary graph in Fig. 1B, BK acutely inhibits ENaC P_o. The mean P_o was 0.47 ± 0.09 and 0.15 ± 0.04 (n = 8, N = 7 mice) in the control and during treatment with BK, respectively. We next tested whether BK actions on ENaC P_o are reversible by perfusing the recording chamber with regular solution. It is not always feasible (due to the complexity of the experimental approach) to obtain the reversal phase in all cases. However, we were successful to reverse the effect of BK in three experiments. The mean P_o following BK washout was 0.44 ± 0.14 (Fig. 1B). Therefore, the results on Fig. 1 are consistent with BK acutely inhibiting ENaC activity in a reversible manner. Importantly, this is the first direct observation of BK regulation of ENaC gating properties in native ASDN.

Fig. 1.

Bradykinin (BK) acutely decreases epithelial sodium channel (ENaC) open probability (P_o) in freshly isolated split-opened aldosterone-sensitive distal nephrons (ASDNs). A: representative continuous current trace from a cell-attached patch containing a single ENaC in the control, under addition of 500 nM BK, and following washout of BK with regular bath solution. This patch was held at a test potential of V_h = −V_p = −60 mV. Areas control (1), after BK treatment (2), and washout (3) are shown below at an expanded time scale. Inward Li⁺ current is downward. Dashed lines indicate the respective current state with a c denoting the closed state. B: summary graph of ENaC P_o changes in response to BK and following washing out from paired patch-clamp experiments similar to that shown in A. *Significant decrease vs. control.

We also probed the dose dependency of BK regulation of ENaC. Figure 2A shows the summary graph of changes in ENaC P_o in response to application of 100 nM BK. In this case, BK causes a smaller (compared with 500 nM) but significant decrease in ENaC P_o. The mean P_o was 0.59 ± 0.09 and 0.31 ± 0.09 (n = 4, N = 4 mice) in the control and after treatment with BK, respectively. Importantly, this effect can also be reversed upon BK washout. The mean P_o was 0.54 ± 0.08 after washout (Fig. 2A). In contrast, 1 nM BK has little effect on ENaC activity (Fig. 2B). The mean P_o was 0.57 ± 0.08 and 0.55 ± 0.08 (n = 4, N = 4 mice). Figure 2C summarizes the dose dependency of BK regulation of ENaC P_o. These results are consistent with BK levels over 1 nM effectively inhibiting ENaC P_o in the ASDN. Importantly, these concentrations are similar to those reported for the renal cortex interstitium (62).

Fig. 2.

Dose dependency of BK regulation of ENaC in split-opened ASDNs. Summary graphs of ENaC P_o changes in response to 100 nM (A) and 1 nM (B) BK and following washing out from paired patch-clamp experiments similar to that shown on Fig. 1. C: inhibition of ENaC P_o in response to application of different concentrations of BK. For each individual experiment, ENaC P_o after corresponding BK treatment was normalized to that for the control condition. *Significant decrease vs. control.

We next determined which BK receptor (i.e., B1 or B2) mediates BK actions on ENaC. Figure 3A contains a representative paired experiment monitoring ENaC activity before, after stimulation of B1 receptors with Lys-Des-Arg⁹-BK (500 nM) and following application of BK (500 nM). Activation of B1 receptors has little effect on ENaC activity (Fig. 3B). Importantly, BK inhibits ENaC P_o even in the presence of Lys-Des-Arg⁹-BK (Fig. 3, A and B). The mean P_o was 0.46 ± 0.03, 0.38 ± 0.08, and 0.07 ± 0.04 (n = 5, N = 4 mice) in the control, after stimulation of B1 receptors with Lys-Des-Arg⁹-BK, and following BK treatment in continued presence of the B1 agonist, respectively. These results are consistent with B1 receptors having a minor role in transducing BK signal to ENaC. In contrast, inhibition of B2 receptors with 500 nM HOE-140 (icatibant) abolishes BK-mediated decreases in ENaC activity as is clear from the representative paired experiment in Fig. 3C and the summary graph in Fig. 3D. The mean P_o was 0.37 ± 0.07, 0.38 ± 0.07, and 0.37 ± 0.06 (n = 5, N = 5 mice) in the control, after HOE-140, and following BK in the continued presence of the B2 antagonist. Therefore, we concluded that B2 rather than B1 receptors play a dominant role in regulation of ENaC by BK.

Fig. 3.

BK inhibits ENaC activity via stimulation of B2 receptors. A: representative continuous current trace from a cell-attached patch monitoring ENaC activity in the control, after stimulation of B1 receptors with a selective agonist, 500 nM Lys-des-Arg⁹-BK, and following application of 500 nM BK in the continued presence of the B1 agonist. Areas control (1), after Lys-des-Arg⁹-BK (2), and following BK (3) are shown below with an expanded scale. All other conditions are identical to those described in Fig. 1A. B: summary graph of ENaC P_o changes in response to the B1 agonist and following BK treatment from paired patch-clamp experiments similar to those shown in A. *Significant decrease vs. control. C: representative continuous current trace from a cell-attached patch monitoring ENaC activity in the control, after inhibition of B2 receptors with a selective antagonist, 500 nM HOE-140, and following application of 500 nM BK in the continued presence of the B2 antagonist. Areas control (1), after HOE-140 (2), and following BK (3) are shown below with an expanded scale. D: summary graph of ENaC P_o changes in response to the B2 antagonist and following BK treatment from paired patch-clamp experiments similar to those shown in C.

It is generally accepted that B2 receptors are coupled to both G_q/11 and G_i leading to stimulation of PLC and depletion of cyclic AMP (cAMP) levels, respectively. To determine which of these signaling pathways has a role in regulation of ENaC by BK, we first treated split-opened ASDNs with nonhydrolysable cell-permeable cAMP analog 8-cpt-cAMP (20 μM). This maneuver is known to saturate cAMP levels, thus, preventing a possible signaling via G_i. 8-cpt-cAMP moderately increases ENaC P_o (Fig. 4, A and B). This is consistent with the idea previously reported by us that vasopressin via V2R-cAMP-PKA pathway increases ENaC activity (8). However, saturation of cAMP levels with 8-cpt-CAMP has little effect on BK-mediated decreases in ENaC activity as is clear from the representative paired experiment on Fig. 4A and the summary graph on Fig. 4B. The mean P_o was 0.35 ± 0.06, 0.50 ± 0.11, and 0.09 ± 0.02 (n = 3, N = 3 mice) in the control, after saturating cAMP levels with 8-cpt-cAMP, and following application of BK in the continued presence of the former, respectively.

Fig. 4.

BK decreases in ENaC P_o via stimulation of G_q/11 but not G_i cascade. A: representative continuous current trace from a cell-attached patch monitoring ENaC activity in the control, after saturation of cellular cAMP levels with 20 μM 8-cpt-cAMP, and following application of 500 nM BK in the continued presence of the former. All other conditions are identical to those described on Fig. 1A. B: summary graph of ENaC P_o changes in response to 8-cpt-cAMP and following BK treatment from paired patch-clamp experiments similar to those shown in A. *Significant decrease vs. control. C: summary graphs of ENaC P_o changes in the control, in response to 500 nM BK and following washout in the presence of G_q/11 inhibition with Gp antagonist 2A (10 μM).

These results suggest a role for G_q/11 in BK signaling pathway to ENaC. To further test this, we next inhibited G_q/11 with Gp antagonist 2A (10 μM). This agent penetrates poorly across the plasma membrane. Thus, freshly isolated ASDNs were incubated for at least 1 h and then split-opened in the presence of Gp antagonist 2A. As summarized in Fig. 4C, BK (500 nM) had a minor effect on ENaC P_o in the presence of G_q/11 inhibition. The mean P_o was 0.37 ± 0.12, 0.35 ± 0.11, and 0.34 ± 0.12 (n = 3, N = 3 mice) in the control, during BK treatment and following washout, respectively. These results suggest that G_q/11 rather than G_i plays a role in BK signal to ENaC.

Activation of G_q/11 is known to stimulate PLC. Thus, we next examined whether PLC is necessary for BK regulation of ENaC activity in the ASDN. Inhibition of PLC with U73122 (10 μM) abolishes BK actions on ENaC activity (Fig. 5, A and B). The mean P_o was 0.42 ± 0.12, 0.59 ± 0.09, and 0.59 ± 0.11 (n = 4, N = 4 mice) in the control, under treatment with U73122, and following application of BK in the continued presence of the PLC inhibitor, respectively. We concluded that activation of B2 receptors in response to BK stimulates G_q/11-PLC pathway to acutely inhibit ENaC P_o.

Fig. 5.

Inhibition of phospholipase C (PLC) abolished BK actions on ENaC. A: representative continuous current trace from a cell-attached patch containing a single ENaC in the control, after inhibition of PLC with 10 μM U73122, and following application of 500 nM BK in the continued presence of the PLC inhibitor. Areas control (1), after U73122 (2), and following BK (3) are shown below with an expanded scale. All other conditions are identical to those described on Fig. 1A. B: summary graph of ENaC P_o changes in response to PLC inhibition and following BK treatment from paired patch-clamp experiments similar to those shown in A.

Activation of PLC leads to PI(4,5)P₂ hydrolysis and elevation of [Ca²⁺]_i due to Ca²⁺ release from the endoplasmic reticulum (ER). To probe whether changes in PI(4,5)P₂ levels play a role in acute regulation of ENaC by BK, we next monitored PI(4,5)P₂ metabolism in mpkCCD_c14 cells using a fluorescent PI(4,5)P₂ reporter, the PH domain of PLC-δ1 fused to GFP during BK signaling with TIRF microscopy. Figure 6A contains representative TIRF micrographs of GFP emission from the apical plasma membrane of polarized mpkCCD_c14 cells before (1), 5 min (2), and 15 min (3) after stimulation with BK. As is clear from these micrographs and the summary graph below, BK rapidly decreases the apical PI(4,5)P₂ levels within several minutes of stimulation. In contrast, vehicle treatment failed to affect apical PI(4,5)P₂ levels (Fig. 6A).

Fig. 6.

Decreases in the apical PI(4.5)P₂ levels parallel with decreases in ENaC activity in response to BK in principal cells. A, top: fluorescence micrographs of emission from the green fluorescent protein (GFP)-PLC-δ1-PH PI(4,5)P₂ reporter in the apical plasma membrane of mpkCCD_c14 cells within a confluent monolayer before (1), and 5 (2), and 15 (3) min after treatment with 500 nM BK. Emissions were collected in a paired manner using total internal reflection fluorescence (TIRF) microscopy. Bottom: time courses of relative PI(4,5)P₂ level changes in the apical plasma membrane after addition of vehicle (black squares) and BK (gray circles). All reagents were added at the beginning of the experiments. All emissions are normalized to those at T = 0 min. B, top: representative continuous current trace from a cell-attached patch on the apical plasma membrane of mpkCCD_c14 cell containing a single ENaC in the control, after treatment with BK, and following inhibition of B2 receptors with HOE-140 in the continued presence of BK. Areas of the control (1), during BK treatment (2), and following HOE-140 application (3) are shown below at an expanded time scale. Bottom: summary graph of changes in ENaC activity (NP_o) in the control, during BK application, and following HOE-140 treatment from paired patch-clamp experiments similar to those shown above. *Significant decrease vs. control.

We (44, 45) and others (24, 28) previously demonstrated that PI(4,5)P₂ can directly interact with ENaC to modulate channel gating. To test whether decreases in PI(4,5)P₂ may account for acute decreases in ENaC P_o in response to BK in these cells, we next assessed the effect of BK on ENaC activity in polarized mpkCCD_c14 monolayers with patch-clamp electrophysiology. Figure 6B shows the representative paired experiment containing a single ENaC before, during BK treatment and following inhibition of B2 receptors with HOE-140. Similar to native preparations (Fig. 1), BK acutely decreases ENaC P_o in cultured principal cells. The mean NP_o was 0.59 ± 0.10 and 0.11 ± 0.03 (n = 6) in the control and under BK treatment, respectively. Moreover, inhibition of B2 receptors, at least partially, restores ENaC activity. Overall, the results in Fig. 6 suggest that stimulation of BK cascade acutely decreases apical PI(4,5)P₂ levels (Fig. 6A) and inhibits ENaC activity (Fig. 6B) in these cultured principal cells.

PI(4,5)P₂ hydrolysis in response to PLC stimulation leads to IP₃ production and triggers Ca²⁺ release from intracellular stores (namely the ER). To test whether increases of [Ca²⁺]_i in response to BK may affect ENaC activity, we pretreated freshly isolated split-opened ASDNs with thapsigargin (2 μM for 30 min) to inhibit Ca²⁺ pump SERCA. However, thapsigargin pretreatment does not abolish BK-mediated decreases in ENaC activity (Fig. 7, A and B). The mean P_o was 0.59 ± 0.08 and 0.21 ± 0.03 (n = 4, N = 3 mice) in control and following BK application, respectively. We next probed whether direct stimulation of Ca²⁺ release from the ER in response to caffeine has an inhibitory effect on ENaC activity. Simply put, can we recapitulate the effect of BK on ENaC by directly stimulating Ca²⁺ release from intracellular stores? Figure 7C contains a representative paired experiment containing a single ENaC before and after treatment with 10 mM caffeine. As is clear from the representative experiment and the summary graph on Fig. 7D, caffeine has little effect on ENaC P_o (0.52 ± 0.10 vs. 0.49 ± 10, n = 6, N = 4 mice). The results on Fig. 7 are consistent with a minor role for elevations in [Ca²⁺]_i in regulation of ENaC activity upon BK stimulation.

Fig. 7.

BK inhibits ENaC P_o independently of Ca²⁺ release from intracellular stores. A: representative continuous current trace from a cell-attached patch containing 2 ENaCs in the control and during application of 500 nM BK. For these experiments, split-opened ASDNs were pretreated with 2 μM thapsigargin to inhibit Ca²⁺ pump SERCA in the endoplasmic reticulum. Areas of control (1) and after BK (2) are shown below with an expanded scale. All other conditions are identical to those described on Fig. 1A. B: summary graph of ENaC P_o changes in response to BK after pretreatment with thapsigargin from paired patch-clamp experiments similar to those shown in A. *Significant decrease vs. control. C: representative continuous current trace from a cell-attached patch containing a single ENaC in the control and during stimulation of intracellular Ca²⁺ release with 10 mM caffeine. Areas of control (1) and after caffeine (2) are shown below with an expanded scale. All other conditions are identical to those described in Fig. 1A. D: summary graph of ENaC P_o changes in response to caffeine from paired patch-clamp experiments similar those shown in C.

Finally, to test whether caffeine raises [Ca²⁺]_i to a similar extent as BK, we next directly monitored changes in [Ca²⁺]_i in freshly isolated split-opened murine ASDNs upon loading with Fura-2. Figure 8A contains the average time courses of elevations in [Ca²⁺]_i in response to application of BK (500 nM) and caffeine (10 mM). We also quantified the effect of purinergic stimulation (ATP, 10 μM) on [Ca²⁺]_i. As have been reported previously by us (41–43) and others (24, 28), ATP targets luminal G protein-coupled P2Y₂ receptors with subsequent activation of G_q/11 signaling pathway to modulate ENaC activity in the ASDN. As documented in Fig. 8A and summarized in Fig. 8B, activation of G_q/11 signaling cascade with both BK and ATP, and direct stimulation of Ca²⁺ release from the ER in response to caffeine, causes prominent increase of the cytosolic [Ca²⁺] to a similar extent. Despite this, the functional consequences of these agents on ENaC activity are drastically different. As summarized in Fig. 8C only BK and ATP, but not caffeine, markedly decrease ENaC activity.

Fig. 8.

Elevations in [Ca²⁺]_i do not play a role in BK-mediated decreases of ENaC P_o. A: average time courses of relative elevations in [Ca²⁺]_i (F₃₄₀/F₃₈₀ ratio) in a single cell within freshly isolated split-opened murine ASDNs loaded with Fura-2 in response to 500 nM BK (left), 10 mM caffeine (middle), and 10 μM ATP (right). At least 6 different ASDNs were evaluated for each group. Solid and open bars denote periods of agonist application and washout, respectively. B: summary graph of F₃₄₀/F₃₈₀ peak magnitude in response to BK, caffeine, and ATP, respectively. C: summary graph of relative ENaC activity after treatment with BK, caffeine, and ATP, respectively. ENaC activity was normalized to the corresponding values before treatment. *Significant change vs. BK.

Overall, we concluded that depletion of the apical PI(4,5)P₂ levels rather than subsequent elevations of [Ca²⁺]_i plays a dominant role in the acute BK-mediated decreases in ENaC P_o in native ASDNs.

DISCUSSION

The major finding of this study is that BK acutely and reversibly decreases ENaC activity by affecting channel P_o in native principal cells. This is the first direct demonstration that renal kinins decrease Na⁺ reabsorption by affecting ENaC gating properties in ASDNs. Substantial evidence suggests that BK promotes natriuresis, in part, by decreasing Na⁺ reabsorption in the distal part of the renal nephron. BK inhibits the amiloride-sensitive component of conductive Na⁺ uptake in freshly prepared inner medullary collecting duct cell suspensions (76). Moreover, BK causes a reversible 50% inhibition of net sodium reabsorption in perfused rat CCDs (66). Finally, in rats fed a normal-salt diet, inhibition of B2 receptors after intrarenal infusion of HOE-140 decreases fractional Na⁺ excretion by 40% but does not alter glomerular filtration rate and medullary and cortical blood flow (63). Our study provides the mechanism for this BK-induced natriuresis. We posit here that renal kinins dynamically adjust the level of renal Na⁺ excretion by affecting ENaC P_o.

The actual BK levels in the kidney are yet to be firmly established. However, the concentration of BK in tissues, including lung, adrenal gland, kidney etc, is believed to be much higher than that found in the circulation (9). A study using a microdialysis technique reports that renal interstitial fluid BK levels in the rat kidney are in the 10- to 100-nM range and these values are higher in the cortex than those in the medulla (62). We found that 100 nM BK acutely inhibits ENaC P_o in the murine ASDN (Fig. 2). This suggests that physiologically relevant concentrations of BK are sufficient to regulate ENaC in its native environment.

Tomita et al. (65, 66) found that BK produces a reversible inhibition of Na⁺ and Cl⁻ transport in perfused rat CCD via an electroneutral mechanism. In contrast, our study provides direct evidence that BK inhibits ENaC activity in split-opened murine ASDNs. This argues that BK inhibits Na⁺ reabsorption at this site via an electrogenic pathway. Interestingly, changes in ENaC activity do not always occur in parallel with changes in trans-epithelial membrane potential (V_te) in perfused CCDs. Thus, increases in tubular flow enhance ENaC-mediated Na⁺ reabsorption without changes in V_te even under conditions where apical K⁺ secretion is inhibited (51). Therefore, the proposed electroneutral mechanism of BK inhibition of NaCl transport (65, 66) does not disqualify ENaC as a possible target of this regulation as suggested here. We have to admit, though, that BK effectively inhibited NaCl transport only from the basolateral side in the perfused CCD from rat (65, 66). In contrast, our study favors the possibility that BK downregulates ENaC P_o from the apical side. We are not certain whether difference in species (rats vs. mice) or in animals preconditioning (prolonged deoxycorticosterone injection vs. low-Na⁺ diet) may contribute to this difference.

KKS is thought to play an important role in controlling systemic blood pressure (1, 32, 68). It appears that all elements of the KKS contribute to this control. Several studies found a correlation between lower urinary kallikrein levels and elevated blood pressure in humans with essential hypertension and in animal hypertensive models (34–36, 46). Conversely, hypertensive mice and rats chronically or transiently overexpressing the human TK gene, which encodes kallikrein, develop hypotension (61, 73). Brown-Norway Katholiek kininogen-deficient rats become hypertensive when put on high-salt diet (32). Finally, mice lacking B2 receptors also develop salt-sensitive hypertension (1, 2). Further studies are required to define pathophysiological consequences of KKS disruption on ENaC-mediated sodium reabsorption particularly upon elevated salt intake.

We found that B2 but not B1 receptors mediate BK actions on ENaC. B2 receptors are shown to be preferentially expressed in the distal nephron including cortical and medullary collecting duct (47, 59). Here, B2 receptors are localized to both apical and basolateral membranes (13, 14). Thus, locally generated kinins from both luminal and interstitial sides are able to stimulate B2 receptors. We cannot precisely discriminate between BK actions from either side of the cell with our experimental approach (split-opened ASDNs). However, we observed a marked BK-stimulated PI(4,5)P₂ hydrolysis as a reflection of G_q/11-PLC activation on the apical side of principal cells with TIRF microscopy (Fig. 6).

B2 receptors are mainly coupled to G_q/11 that leads to activation of PLCβ and elevation of [Ca²⁺]_i by the IP₃-dependent mechanism (6, 20). In addition to that, B2 receptors can be coupled to other plasma membrane G proteins, including G_i. This is known to modulate intracellular cAMP levels and protein kinase A (PKA) activity. We found that inhibition of PLC with U73122 abolishes BK-mediated decreases in ENaC activity (Fig. 5). Moreover, direct inhibition of G_q/11 with Gp antagonist 2A disrupts BK signaling to ENaC (Fig. 4C). In contrast, we found a minor role of G_i in BK regulation of ENaC (Fig. 3, A and B). This is consistent with BK inhibition of ENaC in the ASDN via the G_q/11-PLC pathway.

We document here that activation of BK signaling acutely stimulates the apical PI(4,5)P₂ hydrolysis and inhibits ENaC P_o. We (42, 44, 45) and others (24, 28) previously reported that ENaC activity can be directly determined by PI(4,5)P₂ availability. Depletion of PI(4,5)P₂ levels upon stimulation of G protein-coupled receptor signaling or receptor tyrosine kinase signaling rapidly decreases ENaC activity in expression systems (44, 45, 67), cultured epithelial cells (24, 28, 42), and freshly isolated split-opened ASDNs (41). Moreover, putative PI(4,5)P₂ binding sites in the intracellular NH₂ termini of β- and γ-ENaC subunits have been identified (24, 44). Thus, we speculate that decreases in PI(4,5)P₂ levels are critical for acute inhibition of ENaC P_o in response to BK stimulation.

In contrast to PI(4,5)P₂, a role for elevations in [Ca²⁺]_i in regulation of ENaC activity is less clear. Neither ionomycin nor thapsigargin treatment affected the lumen-to-bath sodium flux in perfused rat CCD, although ionomycin caused marked morphological changes (50). In contrast, ionomycin acutely decreased amiloride-sensitive short-circuit current in the toad urinary bladder (27, 74) and ENaC P_o in freshly isolated split-opened rat CCDs (40). Interestingly, the same study reports that manipulations of [Ca²⁺] had little effect on ENaC P_o in excised inside-out patches from freshly isolated rat CCDs (40). Our study provides strong support to the idea that [Ca²⁺]_i has no significant role in regulation of ENaC activity in ASDNs in response to BK stimulation. First, inhibition of Ca²⁺ pump SERCA in the ER with thapsigargin did not prevent BK-mediated decreases in ENaC activity (Fig. 7, A and B). Second, direct stimulation of Ca²⁺ release from the ER with caffeine did not inhibit ENaC activity (Fig. 7, C and D). Importantly, BK and caffeine elicited a similar rise in [Ca²⁺]_i in split-opened ASDNs as was confirmed using the Ca²⁺ sensor FURA-2 (Fig. 8). Interestingly, ENaC P_o can be acutely inhibited upon stimulation of P2Y₂ receptors, which also leads to a rapid PI(4,5)P₂ hydrolysis. As we reported previously, ATP acutely inhibits ENaC activity via P2Y₂-PLC-PI(4,5)P₂ pathway (41, 42). Therefore, our data suggest that changes in PI(4,5)P₂ levels rather than subsequent elevations in [Ca²⁺]_i play a role in BK-mediated inhibition of ENaC. The apparent controversy with other studies where ionomycin inhibited Na⁺ reabsorption and ENaC activity can be explained by the fact that a robust Ca²⁺ flux into a cell from the bathing media in response to ionomycin is known to stimulate PLC leading to rapid PI(4,5)P₂ hydrolysis (48, 71). In contrast, ionomycin fails to stimulate PLC and, thus, has no effect on the plasma membrane PI(4,5)P₂ levels in Ca²⁺-free medium (71). Therefore, the ionomycin-mediated decreases in ENaC activity may reflect a PI(4,5)P₂ dependency rather than Ca²⁺ dependency.

In conclusion, we found that ENaC-mediated Na⁺ reabsorption in the mammalian ASDN is directly controlled by kinins, such as BK. The signaling pathway involves stimulation of B2-G_q/11-PLC and PI(4,5)P₂ hydrolysis but not [Ca²⁺]_i. This does not exclude a possibility that elevation of [Ca²⁺]_i may trigger stimulation of Ca²⁺-dependent NOS, and prostaglandin release, as it occurs for BK-mediated vasodilation in endothelial cells (30), to further affect ENaC activity in a more prolonged time scale. Indeed, both signaling pathways are proposed to have an important role in regulation of sodium reabsorption in the ASDN (7, 57). Future studies are necessary to carefully examine whether these signaling pathways further contribute to the inhibitory effects of prolonged BK stimulation on ENaC activity in addition to the acute regulation reported here.

GRANTS

This research was supported by the American Heart Association Grant SDG2230391 (to O. Pochynyuk) and the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-70950 (to R. G. O'Neil).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).