Epoxyeicosatrienoic acid metabolites inhibit Kir4.1/Kir5.1 in the distal convoluted tubule

Abstract

Cytochrome P-450 (Cyp) epoxygenase-dependent metabolites of arachidonic acid (AA) have been shown to inhibit renal Na⁺ transport, and inhibition of Cyp-epoxygenase is associated with salt-sensitive hypertension. We used the patch-clamp technique to examine whether Cyp-epoxygenase-dependent AA metabolites inhibited the basolateral 40-pS K⁺ channel (Kir4.1/Kir5.1) in the distal convoluted tubule (DCT). Application of AA inhibited the basolateral 40-pS K⁺ channel in the DCT. The inhibitory effect of AA on the 40-pS K⁺ channel was specific because neither linoleic nor oleic acid was able to mimic the effect of AA on the K⁺ channel. Inhibition of Cyp-monooxygenase with N-methylsulfonyl-12,12-dibromododec-11-enamide or inhibition of cyclooxygenase with indomethacin failed to abolish the inhibitory effect of AA on the 40-pS K⁺ channel. However, the inhibition of Cyp-epoxygenase with N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide abolished the effect of AA on the 40-pS K⁺ channel in the DCT. Moreover, addition of either 11,12-epoxyeicosatrienoic acid (EET) or 14,15-EET also inhibited the 40-pS K⁺ channel in the DCT. Whole cell recording demonstrated that application of AA decreased, whereas N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide treatment increased, Ba²⁺-sensitive K⁺ currents in the DCT. Finally, application of 14,15-EET but not AA was able to inhibit the basolateral 40-pS K⁺ channel in the DCT of Cyp2c44^−/− mice. We conclude that Cyp-epoxygenase-dependent AA metabolites inhibit the basolateral Kir4.1/Kir5.1 in the DCT and that Cyp2c44-epoxygenase plays a role in the regulation of the basolateral K⁺ channel in the mouse DCT.

INTRODUCTION

Cytochrome P-450 (Cyp)-epoxygenase plays an important role in the regulation of renal Na⁺ transport (5, 11, 21, 47). Previous studies have demonstrated that global disruption of Cyp2c44, a major epoxygenase in renal tubules (7), caused salt-sensitive hypertension (5, 21). Increased dietary Na⁺ intake significantly augmented mRNA expression of Cyp2c44 in the distal convoluted tubule (DCT) (40). The DCT is responsible for the reabsorption of 5–9% of filtered Na⁺ load and is the target for thiazide diuretics (9, 10, 15, 22). The DCT is generally divided into the early portion (DCT1) and the late portion (DCT2). While thiazide-sensitive Na⁺-Cl⁻ cotransporter (NCC) is expressed in the apical membrane of both DCT1 and DCT2, channel activity of the renal outer medullary K⁺ channel (ROMK) and epithelial Na⁺ channel (ENaC) is only detected in the apical membrane of DCT2 (1, 9, 35). Reabsorption of NaCl in the DCT1 is a two-step process: Na⁺ and Cl⁻ enter the cells across the apical membrane through NCC and Na⁺ is then pumped out of the cell through basolateral Na⁺-K⁺-ATPase while Cl⁻ exits the cell along its electrochemical gradient by basolateral Cl⁻ channels (ClC-kb) or the K⁺-Cl⁻ cotransporter (18, 20, 34). In the DCT2, Na⁺ enters the cell across the apical membrane through both NCC and ENaC (17, 30). However, in both DCT1 and DCT2, Kir4.1 (Kcnj10)/Kir5.1 (Kcnj16) heterotetramer (a 40-pS inwardly rectifying K⁺ channel) is the only type of K⁺ channel in the basolateral membrane and determines basolateral K⁺ conductance, thereby controlling NCC expression/function (24, 45). The renal phenotype of loss-of-function mutations of Kir4.1 is reminiscent of Gitelman’s syndrome, characterized by salt wasting and hypokalemia, indicating the critical role of Kir4.1 in sustaining the membrane transport in the DCT (3). Because Kir4.1/Kir5.1 plays a key role in regulating membrane transport in the DCT (31), factors affecting Kir4.1/Kir5.1 activity should have a profound effect on both Na⁺ and K⁺ transport in the DCT. Since Cyp-epoxygenase is also expressed in the DCT and its expression is stimulated by high dietary Na⁺ intake (40), it raises the possibility that Cyp-epoxygenase may be involved in the regulation of Kir4.1/Kir5.1 in the DCT. Thus, the aim of the present study was to test whether Cyp-epoxygenase-dependent metabolites of arachidonic acid (AA) play a role in regulating basolateral Kir4.1/Kir5.1 in the DCT.

METHODS

Preparation of DCT.

Male isogenic Cyp2c44^−/− and Cyp2c44^+/+ (wild-type) mice were obtained from Dr. Jorge Capdevilla’s laboratory at Vanderbilt University and were bred at New York Medical College (5). We also purchased 129SvE mice (black Agouti coat), a substrain of 129SvJ mice, from Taconic Farms (Germantown, NY), isogenic with the mice from Vanderbilt University. Mice were kept on a normal diet and had free access to water. We perfused the left kidney with 5 mL collagenase (Worthington CLS II, 1 mg/1 mL) containing L-15 medium (Life Technologies) immediately after mice were euthanized by cervical dislocation. The renal cortex was cut into small pieces after removal of the kidney, and the tissues were then incubated in collagenase-containing L-15 medium at 37°C for 45–60 min. After being rinsed with L-15 medium, the DCTs were dissected in ice-cold media. The isolated DCTs were placed on a coverglass coated with polylysine (Sigma, St. Louis, MO) for patch-clamp experiments. The DCTs were superfused with HEPES-buffered solution containing (in mM) 140 NaCl, 5 KCl, 1.8 MgCl₂, 1.8 CaCl₂, and 10 HEPES (pH 7.4) at room temperature for the single channel recording. All animal procedures were approved by the Institutional Animal Care and Use Committee of New York Medical College.

Patch-clamp experiments.

We used borosilicate glass (1.7-mm outer diameter) to make patch-clamp pipettes, and the pipettes were pulled with a Narishige electrode puller. The pipette had a resistance of 2–4 MΩ when filled with 140 mM KCl. For the single channel recording, we used an Axon 200B patch-clamp amplifier and Axon interface. Currents were low-pass filtered at 1 kHz and digitized by an Axon interface with a 4-kHz sampling rate (Digidata 1440A). Data were analyzed using pClamp software system 9.0 (Axon). The pipette solution for the single channel recording was composed of 140 mM KCl, 1.8 mM MgCl₂, and 10 mM HEPES (pH 7.4), and the bath solution contained 140 mM Na⁺ and 5 mM KCl as described above. Currents were low-pass filtered at 1 kHz and digitized by an Axon interface. Data were analyzed using pClamp software system 10 (Axon). Channel activity, defined as NP_o [a product of channel number (N) and open probability (P_o)], was calculated from data samples of 60-s duration in the steady state as follows: NP_o = Σ (t₁ + 2t₂ +…it_i), where t_i is the fractional open time spent at each of the observed current levels. All patch-clamp experiments were performed in DCT1.

Whole cell recording.

Whole cell patch-clamp experiments were performed in DCT1. An Axon 200A amplifier was used for the measurement of Ba²⁺-sensitive K⁺ currents. For the measurement of whole cell Ba²⁺-sensitive K⁺ currents, the tip of the pipette was filled with pipette solution containing (in mM) 140 KCl, 2 MgCl₂, 1 EGTA, and 10 HEPES (pH 7.4). The pipette was then back filled with pipette solution containing amphotericin B (20 μg/0.1 mL). The bath solution contained (in mM) 140 KCl, 2 MgCl₂, 1.8 CaCl₂, and 10 HEPES (pH 7.4). After formation of a high-resistance seal (>2 GΩ), membrane capacitance was monitored until the whole cell patch configuration was formed. Currents were low-pass filtered at 1 kHz and digitized by an Axon interface with a 4-kHz sampling rate (Digidata 1440A). Data were analyzed using pClamp software system 9.0 (Axon).

Experimental materials and statistics.

AA was obtained from Nu-Check (Elysian, MN), and indomethacin, 5,8,11,14-eicosatetraenoic acid (ETYA), linoleic acid, and oleic acid were purchased from Sigma. We purchased 8,9-epoxyeicosatrienoic acid (EET), 11,12-EET, and 14,15-EET from BioMol. N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) were synthesized in Dr. J. Falck’s laboratory (Southwestern Medical Center, Dallas, TX). Collagenase type 2 was purchased from Worthington (Lakewood, NJ). Data are shown as means ± SE, and Student’s t test or one-way ANOVA was used to determine the statistical significance. If the P value was <0.05, the difference was considered to be significant.

RESULTS

We first used the patch-clamp technique to examine the effect of AA on the basolateral 40-pS K⁺ channel (a Kir4.1/Kir5.1 heterotetramer) in the DCT. Figure 1A shows a single channel recording demonstrating the effect of 5 μM AA on the basolateral 40-pS K⁺ channel in the DCT in a cell-attached patch. It is apparent that 5 μM AA inhibited basolateral 40-pS K⁺ channel activity, as evidenced by the fact that channel P_o decreased progressively, although the current amplitude was only modestly decreased (~10–15%). Results from five experiments (patches) are shown in Fig. 1B, demonstrating that 5 μM AA decreased K⁺ channel activity defined by NP_o from 1.82 ± 0.25 to 0.53 ± 0.11. The effect of AA on the basolateral K⁺ channel in the DCT was specific since application of 5 μM linoleic acid (1.85 ± 0.2, n = 5) or 5 μM oleic acid (1.75 ± 0.22, n = 5) failed to inhibit the 40-pS K⁺ channel. Moreover, the addition of 5 μM ETYA, an analog of AA, failed to inhibit the 40-pS K⁺ channel (NP_o: 1.72 ± 0.23, n = 5).

Fig. 1.

Arachidonic acid (AA) inhibits the basolateral 40-pS K⁺ channel in the distal convoluted tubule (DCT). A: single channel recording showing the effect of AA (5 μM) on the basolateral 40-pS K⁺ channel in the DCT. The top trace shows the time course of the experiments, and three parts of the trace indicated by a number are extended to show the fast time resolution. Experiments were performed in a cell-attached patch with 140 mM K⁺ in the pipette and 140 mM Na⁺/5 mM K⁺ in the bath. The holding potential was 0 mV. The channel closed level is indicated by the letter C. B: bar graph summarizing the effect of 5 μM AA, linoleic acid, oleic acid, and 5,8,11,14-eicosatetraenoic acid (ETYA) on the basolateral 40-pS K⁺ channel in the DCT. *Significant difference compared with other groups. Experiments were performed in cell-attached patches.

After demonstrating that AA inhibited the basolateral K⁺ channel in the DCT, we next examined whether the inhibitory effect of AA on the 40-pS K⁺ channel was by AA per se or by AA metabolites. Three enzymes, cyclooxygenase, Cyp-monooxygenase, and Cyp-epoxygenase (29), have been shown to metabolize AA in the kidney under physiological conditions. Thus, we first examined whether the inhibitory effect of AA on the K⁺ channel was mediated by Cyp-monooxygenase by testing the effect of AA on the 40-pS K⁺ channel in the DCT treated with DDMS (10 μM) (37). Figure 2A shows a single channel recording made in a cell-attached patch, and Fig. 2B shows the results of seven experiments. Treatment of the DCT with DDMS for 5 min had no significant effect on K⁺ channel activity (control NP_o: 1.78 ± 0.21 and DDMS NP_o: 1.68 ± 0.18). Moreover, application of AA still decreased K⁺ channel activity (NP_o: 0.46 ± 0.1), suggesting that the inhibitory effect of AA was not dependent on Cyp-monooxygenase. We next examined the effect of AA on the basolateral 40-pS K⁺ channel in the DCT treated with indomethacin (5 μM). Results from six experiments are shown in Fig. 2B, demonstrating that treatment of the DCT with indomethacin also had no significant effect on K⁺ channel activity (NP_o: 1.9 ± 0.17). Also, AA was able to inhibit the 40-pS K⁺ channel (NP_o: 0.7 ± 0.15), suggesting that cyclooxygenase was not responsible for the inhibitory effect of AA on the K⁺ channel.

Fig. 2.

Inhibition of cytochrome P-450 (Cyp)-monooxygenase and -cyclooxygenase does not affect arachidonic acid (AA)-induced inhibition of basolateral 40-pS K⁺ channels. A: single channel recording showing the effect of AA (5 μM) on the basolateral 40-pS K⁺ channel in the distal convoluted tubule (DCT) treated with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). The top trace shows the time course of the experiments, and two parts of the trace indicated by a number are extended to show the fast time resolution. Experiments were performed in a cell-attached patch with 140 mM K⁺ in the pipette and 140 mM Na⁺/5 mM K⁺ in the bath. The holding potential was 0 mV. The channel closed level is indicated by the letter C. B: bar graph summarizing the effect of 10 μM DDMS, DDMS+AA, 5 μM indomethacin (Indo), and Indo + AA on the basolateral 40-pS K⁺ channel in the DCT. Experiments were performed in cell-attached patches.

We then examined the effect of AA on the 40-pS K⁺ channel in the DCT treated with MS-PPOH (an inhibitor of Cyp-epoxygenase) (37). Figure 3A shows a channel recording made in a cell-attached patch demonstrating the effect of 5 μM AA on the basolateral K⁺ channel in the presence of 10 μM MS-PPOH, and Fig. 3B shows a bar graph summarizing the results from six experiments. Although inhibition of Cyp-epoxygenase had no significant effect on 40-pS K⁺ channel activity in the DCT (control NP_o: 1.6 ± 0.18 and MS-PPOH NP_o: 1.65 ± 0.18), inhibition of Cyp-epoxygenase was able to abolish the effect of AA on the 40-pS K⁺ channel (NP_o: 1.50 ± 0.16). This strongly suggests the role of Cyp-epoxygenase in mediating the effect of AA on the 40-pS K⁺ channel in the DCT. The notion that Cyp-epoxygenase may mediate the inhibitory effect of AA on the basolateral K⁺ channel was also suggested by the finding that MS-PPOH can partially reverse the inhibitory effect of AA on the 40-pS K⁺ channel. Figure 4 shows a single channel recording demonstrating that application of 5 μM AA inhibited the basolateral 40-pS K⁺ channel in the DCT and decreased NP_o from 1.61 ± 0.12 to 0.42 ± 0.0.06 (n = 4). In contrast, the addition of 10 μM MS-PPOH partially reversed the inhibitory effect of 5 μM AA on the K⁺ channel and increased NP_o to 0.9 ± 0.1.

Fig. 3.

Inhibition of cytochrome P-450 (Cyp)-epoxygenase abolished the effect of arachidonic acid (AA) on the basolateral 40-pS K⁺ channel in the distal convoluted tubule (DCT). A: single channel recording showing the effect of AA (5 μM) on the basolateral 40-pS K⁺ channel in the DCT treated with N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH). The top trace shows the time course of the experiments, and three parts of the trace indicated by a number are extended to show the fast time resolution. Experiments were performed in a cell-attached patch with 140 mM K⁺ in the pipette and 140 mM Na⁺/5 mM K⁺ in the bath. The holding potential was 0 mV. The channel closed level is indicated by the letter C. B: bar graph summarizing the effect of 10 μM MS-PPOH and MS-PPOH + 5 μM AA on the basolateral 40-pS K⁺ channel in the DCT. Experiments were performed in cell-attached patches.

Fig. 4.

Inhibition of cytochrome P-450 (Cyp)-epoxygenase partially reversed the effect of arachidonic acid (AA) on the basolateral 40-pS K⁺ channel in the distal convoluted tubule (DCT). The single channel recording shows that the effect of AA (5 μM) on the basolateral 40-pS K⁺ channel was partially reversed by adding 10 μM N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH) in the DCT. The top trace shows the time course of the experiments, and three parts of the trace indicated by a number are extended to show the fast time resolution. Experiments were performed in a cell-attached patch with 140 mM K⁺ in the pipette and 140 mM Na⁺/5 mM K⁺ in the bath. The holding potential was 0 mV. The channel closed level is indicated by the letter C.

We also used the perforated whole cell recording to examine the effect of AA and MS-PPOH on Ba²⁺-sensitive K⁺ currents in DCT1. Since basolateral Kir4.1/Kir5.1 is the only type of K⁺ channel in DCT1, the whole cell K⁺ conductance is equal to Kir4.1/Kir5.1 activity (8). Figure 5A shows a Ba²⁺-sensitive current trace measured with a ramp protocol from −100 to 100 mV using perforated whole cell recording, and Fig. 5B shows a scatterplot demonstrating the mean value and each data point of a total of six experiments. Application of 5 μM AA for 15–20 min inhibited whole cell Ba²⁺-sensitive K⁺ currents from 1,180 ± 95 pA to 400 ± 60 pA (n = 6). In contrast, application of MS-PPOH for 15–20 min increased whole cell Ba²⁺-sensitive K⁺ currents to 1,860 ± 125 pA (n = 6).

Fig. 5.

N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH) increased, whereas arachidonic acid (AA) decreased, whole cell K⁺ currents in the early distal convoluted tubule (DCT1). A: recording showing whole cell Ba²⁺-sensitive K⁺ currents measured with a ramp protocol from −100 to 100 mV in DCT1 treated with 5 μM AA or 10 μM MS-PPOH for 15–20 min. B: scatterplot summarizing the results of 6 experiments in which whole cell Ba²⁺-sensitive K⁺ currents were measured at −60 mV. The mean value is shown on the left side of each column.

After establishing the role of Cyp-epoxygenase-dependent metabolites of AA in regulating the basolateral 40-pS K⁺ channel in the DCT, we next examined which metabolite was responsible for the effect of AA on the 40-pS K⁺ channel in the DCT. Thus, we used the single channel recording to examine the effect of 100 nM 8,9-EET, 11,12-EET, and 14,15-EET. Figure 6A shows a set of single channel recordings demonstrating the effect of 8,9-EET, 11,12-EET, and 14,15-EET on the basolateral 40-pS K⁺ channel in the DCT (n = 5–7). Figure 6B shows a bar graph summarizing the effect of experiments in which the effect of EET on the 40-pS K⁺ channel in the DCT was examined in cell-attached patches. From inspection of Fig. 6, it is apparent that 11,12-EET and 14,15-EET but not 8,9-EET were able to inhibit the basolateral 40-pS K⁺ channel in the DCT. Application of 100 nM 11,12-EET or 14,15-EET decreased the 40-pS K⁺ channel to 29 ± 3% and 20 ± 3% of the corresponding control, respectively.

Fig. 6.

Epoxyeicosatrienoic acid (EET) inhibits the basolateral 40-pS K⁺ channel in the distal convoluted tubule (DCT). A: set of single channel recordings showing the effect of 100 nM 8,9-EET, 11,12-EET, and 14,15-EET on the basolateral 40-pS K⁺ channel in the DCT. B: bar graph summarizing the effect of 5 μM arachidonic acid (AA; n = 5), 100 nM 11,12-EET (n = 6), 14,15-EET (n = 7), and 8,9-EET (n = 5) on basolateral K⁺ channel activity of the DCT. Normalized channel activity was used as the control value. *Significant difference. C: bar graph summarizing the effect of 5 μM AA and 100 nM 14,15-EET on the basolateral 40-pS K⁺ channel in the DCT of cytochrome P-450 (Cyp)2c44^−/− mice (n = 6).

It is well established that rat CYP2C23 or mouse Cyp2c44 is the predominant Cyp-epoxygenase in the kidney and endothelium and is responsible for converting AA to EET (7). After demonstrating that 11,12-EET or 14,15-EET was able to inhibit the basolateral 40-pS K⁺ channel in the DCT, we then examined the effect of AA (5 μM) or 100 nM 14,15-EET on the 40-pS K⁺ channel of the DCT in Cyp2c44-deficient mice. Results from six experiments are shown in Fig. 6C, demonstrating that the application of AA failed to inhibit K⁺ channel activity (control NP_o: 2.3 ± 0.19 and AA NP_o: 2.2 ± 0.19). However, application of 100 nM 14,15-EET was still able to inhibit the basolateral 40-pS K⁺ channel in the DCT of Cyp2C44-deficient mice and reduced NP_o to 0.52 ± 0.10, suggesting a role of Cyp2c44 in mediating the inhibitory effect of AA on the K⁺ channel in the DCT

DISCUSSION

The main finding of the present study was to demonstrate that Cyp-epoxygenase-dependent AA metabolites inhibit the basolateral 40-pS K⁺ channel in the DCT. This notion is supported by four lines of evidence. First, inhibition of Cyp-epoxygenase, but not cyclooxygenase or Cyp-monoxygenase, abolished the inhibitory effect of AA on the 40-pS K⁺ channel in the DCT. Second, Cyp-epoxygenase-dependent AA metabolites, 11,12-EET and 14,15-EET, were able to mimic the effect of AA and inhibited the basolateral 40-pS K⁺ channel in the DCT. Third, inhibition of Cyp-epoxygenase with MS-PPOH increased K⁺ currents in DCT1. Finally, AA failed to inhibited the 40-pS K⁺ channel in Cyp2C44^−/− mice, but 14,15-EET was still able to inhibit the K⁺ channel in the DCT of Cyp2c44^−/− mice. This strongly suggests that Cyp-epoxygenase activity is involved in the regulation of the basolateral 40-pS K⁺ channel in the DCT.

The basolateral 40-pS K⁺ channel is composed of Kir4.1 and Kir5.1. Although Kir4.1 alone can form a homotetramer (a 20- to 25-pS K⁺ channel) in vivo and in vitro (23, 24, 26), Kir.4.1 prefers interacting with Kir5.1 to form a functional 40-pS heterotetrameric K⁺ channel in the basolateral membrane of the native tissue under physiological conditions (16, 44, 45). Thus, our study suggests that Cyp-epoxygenase-dependent AA metabolites play an important role in the regulation of basolateral Kir4.1/Kir5.1 in the DCT. The DCT is an initial nephron segment of the aldosterone-sensitive distal nephron (ASDN), and the DCT plays an important role in mediating hormone-regulated Na⁺ absorption and in the regulation of renal K⁺ excretion by controlling Na⁺ and fluid volume delivery to the late ASDN. Because Kir4.1/Ki5.1 expression is overlapped with NCC, ENaC, and ROMK along the distal nephron (32), the activity of Kir4.1/Ki5.1 should affect membrane transport in the distal nephron. Indeed, previous experiments performed in kidney-specific Kir4.1 knockout mice have demonstrated that basolateral Kir4.1 channel activity in the DCT determines NCC expression (6, 38). Similar phenotypes have also been found in caveolin-1 knockout mice in which Kir4.1/Ki5.1 activity was inhibited (36). Consequently, the renal phenotype of caveolin-1 knockout mice is similar to Kir4.1 knockout mice, including low-K⁺ conductance in the DCT and low NCC activity. The effect of Kir4.1 on NCC activity is possibly mediated by the Cl⁻-sensitive with-no-lysine kinase and Ste20-proline-alanine-rich kinase pathway (2, 27). Thus, changes in basolateral Kir4.1/Kir5.1 activity should have a profound effect on NCC activity and Na⁺ absorption in the DCT. The finding that 11,12-EET and 14,15-EET inhibit basolateral Kir4.1/Kir5.1 activity in the DCT strongly suggests the role of Cyp-epoxygenase in the inhibition of Na⁺ transport in the DCT. In this regard, previous studies have reported that CYP-epoxygenases play a role in preventing salt-sensitive hypertension in animal models (5, 21).

The CYP-epoxygenases expressed in the mouse kidney include Cyp2c29, Cyp2c38, Cyp2c39, Cyp2c44, and Cyp2J5 (7, 33), and they play a role in the regulation of renal vascular tone and Na⁺ transport (5, 12, 13, 46). However, among the Cyp-epoxygenase family, members of the CYP2C subfamily (in particular, rat CYP2C23 and mouse CYP2C44) are the predominant and functionally relevant kidney epoxygenases (4, 7, 14, 43). They are mainly responsible for the generation of 11,12-EET and 14,15-EET in the kidney (7, 19, 21). Previous studies have demonstrated that kidney expression of CYP2C-epoxygenases was dietary salt sensitive (19, 47). Downregulation of Cyp2c23 has been shown to link to salt- or angiotensin II-induced hypertension (39, 46, 47). Moreover, a large body of evidence indicates that Cyp2C-epoxygenase regulates Na⁺ transport in the ASDN (21, 25, 28, 41). We and others have previously demonstrated that EETs inhibit ENaC and that Cyp2c44-dependent generation of 11,12-EET is mainly responsible for suppressing ENaC in the cortical collecting duct because the disruption of Cyp2c44 significantly increased the basal level of ENaC activity in the cortical collecting duct (21, 25, 28, 41).

The physiological significance of our study is to suggest that in addition to the inhibition of ENaC in the ASDN, Cyp2C-epoxygenase family-dependent AA metabolites may play a role in regulating NCC by targeting Kir4.1/Kir5.1 in the DCT. Indeed, high salt intake has been shown to inhibit basolateral Kir4.1/Kir5.1 activity in the DCT (42). Since high salt intake stimulated expression of Cyp2c23 (a homolog of mouse Cyp2c44) in rats (40), it is possible that Cyp-epoxygenase-dependent metabolites may be involved in inhibiting basolateral Kir4.1/Kir5.1 during high Na⁺ intake. The finding that 14,15-EET but not AA was able to block 40-pS K⁺ channels in the DCT of Cyp2C44^−/− mice strongly suggests that Cyp2C44 is responsible for AA-induced inhibition of 40-pS K⁺ channels in the mouse kidney. Since Kir4.1/Kir5.1 in the DCT determines NCC activity, EET-induced inhibition of basolateral K⁺ channels in the DCT is expected to indirectly reduce NCC activity in response to high salt intake. Further study is required to test the role of EET in mediating the effect of high Na⁺ intake on NCC. We conclude that Cyp-epoxygenase-dependent AA metabolites inhibit basolateral K⁺ channels in the DCT and that Cyp2c44-epoxygenase is mainly responsible for mediating the effect of AA on Kir4.1/Kir5.1 activity in the mouse DCT.

GRANTS

M.-X. Wang’s research is supported by National Natural Science Foundation of China Grants 31660286 and 81900652. L.-J. Wang’s research is supported by National Natural Science Foundation of China Grant 31500939 and UNPYSCT Grant 2016043. Y. Xiao’s research is supported by Education Department of Heilongjiang Grant 2016-KYYWF-0850. W.-H. Wang’s research is supported National Institutes of Health Grant DK-54983.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

W.-H.W. conceived and designed research; M.-X.W., L.-J.W., Y.X., D.-D.Z., and X.-P.D. performed experiments; M.-X.W., L.-J.W., and W.-H.W. analyzed data; M.-X.W., L.-J.W., X.-P.D., and W.-H.W. interpreted results of experiments; M.-X.W., L.-J.W., Y.X., D.-D.Z., and W.-H.W. prepared figures; W.-H.W. drafted manuscript; M.-X.W. and W.-H.W. edited and revised manuscript; M.-X.W., L.-J.W., Y.X., D.-D.Z., X.-P.D., and W.-H.W. approved final version of manuscript.