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Epoxyeicosatrienoic Acid Activates BK Channels in the Cortical Collecting Duct

Peng Sun,* Wen Liu,† Dao-Hong Lin,* Peng Yue,* Rowena Kemp,* Lisa M. Satlin,† and Wen-Hui Wang*

*Department of Pharmacology, New York Medical College, Valhalla, and †Department of Pediatrics, Mount Sinai School of Medicine, New York, New York

ABSTRACT The cortical collecting duct (CCD), which is involved in renal potassium (K) excretion, expresses cyto- chrome P450 (CYP)-epoxygenase. Here, we examined the effect of high dietary K on renal expression of CYP2C23 and CYP2J2 in the rat, as well as the role of CYP-epoxygenase–dependent metabolism of ϩ in the regulation of Ca2 -activated big-conductance K (BK) channels. By Western blot analysis, high dietary K stimulated the expression of CYP2C23 but not CYP2J2 and increased 11,12- (11,12-EET) levels in isolated rat CCD tubules. Application of arachidonic acid increased BK channel activity, and this occurred to a greater extent in rats on a high-K diet compared with a normal-K diet. This effect was unlikely due to arachidonic acid–induced changes in membrane fluidity, because 11,14,17-eicosatrienoic acid did not alter BK channel activity. Inhibiting CYP-epoxyge- nase but not - or CYP-␻-hydroxylase–dependent pathways completely abolished the stimulatory effect of arachidonic acid on BK channel activity. In addition, application of 11,12-EET mimicked the effect of arachidonic acid on BK channel activity, even in the presence of CYP-epoxygenase inhibition. This effect seemed specific to 11,12-EET, because both 8,9- and 14,15-EET failed to stimulate BK channels. Finally, inhibition of CYP-epoxygenase abolished iberiotoxin-sensitive and flow-stimulated but not basal net K secretion in isolated microperfused CCD. In conclusion, high dietary K stimulates the renal CYP-epoxygenase pathway, which plays an important role in activating BK channels and flow- stimulated K secretion in the CCD.

J Am Soc Nephrol 20: 513–523, 2009. doi: 10.1681/ASN.2008040427

The kidney plays a key role in maintaining external renal K secretion under normal conditions.3,10,11 potassium (K) in a normal range despite the varia- Conversely, a large body of evidence has strongly tion of dietary K intake: High K (HK) intake in- indicated that Ca2ϩ-activated BK channels are in- creases and low K intake decreases K secretion in 1 the kidney. The renal K secretion takes place in the Received April 25, 2008. Accepted September 17, 2008. connecting tubule (CT) and initial cortical collect- Published online ahead of print. Publication date available at ing duct (CCD) by a two-step process: K enters the www.jasn.org. cell through the basolateral Na-K-ATPase and P.S. and W.L. contributed equally to this work and should be leaves the cell by the apical K channels. Two types of considered as co-first authors. K channels—ROMK- and Ca2ϩ-activated big-con- R.K.’s and L.S.M.’s laboratories are equal contributors to this ductance K (BK) channels—are expressed in the study. apical membrane of both CT and CCD.2–7 Because Correspondence: Dr. Wen-Hui Wang, Department of Pharma- BK channels have a low open probability and low cology, BSB 537, New York Medical College, Valhalla, NY channel density in the CCD from animals on a nor- 10595. Phone: 914-594-4139; Fax: 914-347-4956; E-mail: mal K (NK) intake,8,9 it is generally accepted that [email protected] ROMK rather than BK channels are responsible for Copyright ᮊ 2009 by the American Society of Nephrology

J Am Soc Nephrol 20: 513–523, 2009 ISSN : 1046-6673/2003-513 513 BASIC RESEARCH www.jasn.org volved in K secretion in the CCD subjecting to high flow rates A B or in animals with increased K intake12–14; however, the mech- 200 * anism by which HK intake stimulates BK channel–dependent 150 K secretion is not understood. NK HK ϩ Ca2 -activated BK channels are also expressed in smooth CYP2C23 100 * muscle cells and endothelial cells and have been shown to be 50 15–17 activated by epoxyeicosatrienoic acid (EET), a product Actin Change of Expression of control) (% generated by (CYP)-epoxygenase–depen- 0 CYP2C23 CYP2J2 dent arachidonic acid (AA) metabolism.18 CYP-epoxygenase– dependent AA metabolites play an important role in the regu- NK HK C lation of Na transport in the CCD.19 We previously demonstrated that 11,12-EET inhibited epithelial Na channels CYP2J2 (ENaC).20 We and others have shown that CYP-epoxygenases such as CYP2C23 and CYP2J2 are expressed in the CCD.21,22 Moreover, CYP2C23 has been shown to be a major isoform of Actin CYP-epoxygenase responsible for 11,12-EET formation in the kidney.23,24 Thus, the aim of this study is to test the hypothesis that HK intake may stimulate CYP-epoxygenase activity and increase EET, which plays a role in stimulating the BK channel Figure 1. (A) Western blot shows the effect of K intake on the activity in the CCD. expression of CYP2C23 and CYP2J2 in renal cortex and outer medulla (mixture) from rats on an NK or HK diet. (B) Bar graph summarizes the changes in CYP2C23 and CYP2J2 expression RESULTS (data are normalized in comparison with actin level). *Significant difference (P Ͻ 0.05). (C) Effect of HK on 11,12-EET level in isolated CCDs. *Significant difference. HK Increases CYP-Epoxygenase The main types of CYP-epoxygenases expressed in the rat CCD are CYP2C23 and CYP2J2.21,22 Because the CCD is responsible involved in mediating K secretion in the late distal tubule in- for K secretion, we examined whether increased dietary K in- cluding connecting tubule (CT) in response to HK intake.13,25 take affected the expression of CYP2C23 and CYP2J2. Figure Thus, we examined whether CYP-epoxygenase–dependent 1A is the Western blot analysis demonstrating the expression AA metabolism regulates BK channels in the CCD. We con- of CYP2C23 and CYP2J2 in the renal cortex and outer medulla firmed the previous finding that BK channel activity was low in (mixture) from rats on an HK diet or an NK diet. Data sum- the CCD from rats on an NK diet and slightly increased in the marized in Figure 1B demonstrate that HK intake significantly CCD of rats on an HK diet2; however, the channel open prob- increased the expression of CYP2C23 by 90 Ϯ 10% (n ϭ 5) but ability of BK was still low under basal conditions. Figure 2A is decreased the expression of CYP2J2 by 40 Ϯ 8% (n ϭ 3). Our a recording showing the BK channel activity in a principal cell previous experiments demonstrated that the CYP2C23 was (PC) of the CCD from rats on an HK diet, and the mean chan- 22 highly expressed in the thick ascending limb and the CCD. nel activity defined by NPo (a product of channel number and Thus, it is conceivable that the expression of CYP2C23 in the open probability) was 0.03 Ϯ 0.01 (n ϭ 6). We then examined CCD should be augmented in rats fed with HK diet. This spec- the effect of AA on BK channels while the channel activity was ulation was confirmed by experiments in which the 11,12-EET continuously monitored in a cell-attached mode. Application concentrations were measured in the isolated CCD tubules of AA (10 ␮M) significantly stimulated BK channel activity Ϯ ϭ from rats on NK or HK diet. Because 11,12-EET accounts for and increased NPo to 0.62 0.20 (n 6). We also studied the Ͼ60% of the total renal EET,15,24 alteration of 11,12-EET effect of AA on BK channels with low concentrations of AA. should reflect the CYP-epoxygenase activity. Figure 1C sum- Figure 2B is a dose-response curve of AA effect on BK channels marizes the results and shows that HK intake significantly in- showing that application of 3 and 5 ␮MAA(n ϭ 5) increased creased 11,12-EET concentration from 0.65 Ϯ 0.10 to 1.70 Ϯ BK channel activity to 0.10 Ϯ 0.04 and 0.30 Ϯ 0.08 (P Ͻ 0.05), 0.25 ng/mg protein (n ϭ 4 rats). Thus, HK intake stimulates respectively. the activity of CYP-epoxygenase and 11,12-EET levels in the To exclude the possibility that the AA effect on BK channels CCD. may be due to nonspecifically changing the membrane fluidity, which has been reported to affect channel activity,26,27 we then AA Activates BK Channels examined the effect of 11,14,17-eicosatrienoic acid (11,14,17- It is well established that CYP-epoxygenase is involved in me- EA) on BK channels in the CCD from rats on HK diet. From tabolizing AA to produce EET, which has been shown to acti- the inspection of Figure 2C, it is apparent that 11,14,17-EA fails vate BK channels in smooth muscle cells.15,17 BK channels are to stimulate BK channels. Data summarized in Figure 2D dem- also expressed in the CCD13,25 and have been suggested to be onstrate that application of 10 ␮M 11,14,17-EA did not alter

514 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 513–523, 2009 www.jasn.org BASIC RESEARCH

A B To demonstrate further that the effect of AA is mediated specifically by CYP-epoxygenase, we C examined the effect of AA on BK channel in the presence of various inhibitors of AA metabolic N P o pathways. There are three types of enzymes— CYP-epoxygenase, cyclooxygenase (COX), and C CYP-␻-hydroxylase—which have been shown to metabolize AA in renal tubules.28,29 To test the role of COX in mediating the effect of AA on BK C channels, we examined the effect of AA in the

D 0.9 presence of indomethacin, an inhibitor of COX. * Application of indomethacin (5 ␮M) alone had

0.6 no significant effect on BK channels, suggesting C that endogenous COX was not involved in regu- NPo C lating the BK channel activity in the CCD (data 0.3 not shown). Moreover, as shown in Figure 4A, inhibiting COX did not abolish the AA-induced 0.0 stimulation (top trace) because application of AA AA still increased BK channel activity from Control Control Ϯ Ϯ ϭ

11,14,17-EA 0.029 0.010 to 0.900 0.170 (n 5; Figure 4B). This indicates that the effect of AA on BK Figure 2. (A) A channel recording showing the effect of AA on BK channels in channels was not medicated by COX-dependent the CCD. The experiments were performed in a cell-attached patch, and the holding potential was 0 mV. The channel closed level is indicated by C and a metabolites. dotted line. The top trace shows the time course of the experiment, and two We also investigated whether the effect of AA ␻ parts of the recording indicated by numbers and a bar are extended to show the on BK channels depends on the CYP- -hydrox- fast time resolution. (B). A dose-response curve of AA effect on BK channels. ylase–dependent AA metabolism. Similar to in- Each point represents a mean value from five to six patches. (C) A recording domethacin, inhibiting CYP-␻-hydroxylase showing the effect of 10 ␮M 11,14,17-EA on BK channels in the CCD. (D) The with 5 ␮M N-methylsulfonyl-12,12-dibromodo- effect of 10 ␮M 11,14,17-EA and AA on BK channel activity. The experiments dec-11-enamide (DDMS), an inhibitor of CYP- were performed in cell-attached patches of the CCD from rats on a HK diet. ␻-hydroxylase,28 had no significant effect on BK Ͻ *Significant difference (P 0.05). The pipette solution was composed of 140 mM channels (data not shown). Moreover, inhibition KCl, 1.8 mM MgCl2, and 5 mM HEPES (pH 7.4). of CYP-␻-hydroxylase did not affect the AA-in- Ϯ Ϯ channel activity (control NPo 0.040 0.010, EA 0.035 0.010; duced stimulation of BK channels in the CCD. Figure 4A, mid- n ϭ 6; P Ͼ 0.05). Thus, it is unlikely that the effect of AA on BK dle, is a channel recording showing that inhibiting CYP-de- channels was due to the alteration of the membrane physical pendent ␻-oxidation with 5 ␮M DDMS failed to abolish the properties. stimulatory effect of AA on BK channels. In the presence of Ϯ DDMS, AA increased BK channel NPo from 0.04 0.01 to Effect of AA Is Mediated by CYP-Epoxygenase– 0.82 Ϯ 0.14 (n ϭ 6; P Ͻ 0.01). Thus, the CYP-␻-hydroxylase Dependent Pathway pathway was not involved in the activation of BK channels in After demonstrating that AA specifically stimulated BK chan- CCD. We then examined the role of CYP-epoxygenase in me- nels in the CCD, we examined whether the effect of AA on BK diating the effect of AA on BK channels in the CCD (Figure 4A, channels was mediated by the CYP-epoxygenase metabolic bottom). Although inhibiting CYP-epoxygenase with pathway. As demonstrated in Figure 1, CYP-epoxygenase ac- N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS- tivity is lower in the CCDs from rats on a NK diet than that on PPOH), an inhibitor of CYP-epoxygenase,30 had no significant Ϯ a HK diet. Thus, we speculated that the stimulatory effect of AA effect on BK channel activity (control NPo 0.029 0.007, MS- should be smaller in rats on an NK diet than that on an HK diet PPOH 0.030 Ϯ 0.009; n ϭ 4), AA-induced stimulation of BK if CYP-epoxygenase–dependent AA metabolism is responsible channels was absent in the presence of 5 ␮M MS-PPOH (Fig- for the AA-induced stimulation of BK channel activity. Figure ure 4B). This strongly suggests that the stimulatory effect is 3A is a channel recording showing that application of 10 ␮M mediated by the CYP-epoxygenase–dependent metabolic AA also increased the BK channel activity; however, the stim- pathway. ulatory effect of AA was significantly smaller in rats on an NK than that on an HK diet. Data summarized in Figure 3B dem- 11,12-EET Activates BK Channels ␮ Ϯ onstrate that 10 M AA increased NPo from 0.01 0.05 to Because 11,12-EET is a major product of CYP-epoxygenase– 0.10 Ϯ 0.10 (n ϭ 6), a value that is significantly smaller than dependent AA metabolism,24 we examined the effect of 11,12- 0.62 Ϯ 0.20 observed in the CCD from HK-adapted rats. EET on BK channels. Figure 5 is a channel recording showing

J Am Soc Nephrol 20: 513–523, 2009 EET Activates BK Channels in the CCD 515 BASIC RESEARCH www.jasn.org

ABfects of 8,9- and 14,15-EET on BK channels. From inspection of Figure 6A, it is apparent that 8,9- C HK EET had no effect on BK channels, whereas 100 0.9 * # nM 11,12-EET activated the BK channels in the 1 2 same patch. Also, 14,15-EET failed to activate BK AA 20S channels (Figure 6B). Data summarized in Figure 5pA 0.6 1. Control 6C show that neither 8,9-EET (control 0.027 Ϯ C Ϯ NPo NK 0.003; 8,9-EET 0.029 0.003) nor 14,15-EET had significant effect on BK channels (control 0.03 Ϯ 0.3 0.005; 14,15-EET 0.03 Ϯ 0.004). In contrast, ad- 2. 10 µMAA C dition of 11,12-EET increased BK channel activity * from 0.030 Ϯ 0.006 to 0.890 Ϯ 0.300 (n ϭ 6). 0.0 Thus, AA-induced stimulation of BK channels is

AA mediated by 11,12-EET in the CCD. This notion is 0.8S AA 5pA Control further supported by the observation that 11,12- Control EET still activated BK channels in the presence of Figure 3. (A) A channel recording showing the effect of 10 ␮MAAonBK MS-PPOH, which blocked the effect of AA on BK channels in the CCD from rats on NK diet. The top trace is the time course of channels at the same patch. From inspection of the experiment, and two parts of the recording indicated by numbers are Figure 7, it is apparent that the stimulatory effect extended to show the fast time resolution. The channel closed level is indicated of AA on BK channels was absent in the CCD by C, and the holding potential was 0 mV. An arrow indicates the addition of treated with MS-PPOH; however, in the continu- AA. (B) A bar graph summarizes the effect of AA on BK channels in the CCD ous presence of MS-PPOH, addition of 100 nM from rats on NK or HK diet, respectively. *Significant difference between AA 11,12-EET activated BK channels and increased and corresponding control group; #significance between HK and NK groups. Ϯ Ϯ ϭ NPo from 0.030 0.009 to 0.840 0.200 (n 5).

A Indomethacin B Inhibition of CYP-Epoxygenase Blocked the C BK Channel–Mediated Flow-Stimulated Net K Secretion in the CCD We then examined the role of CYP-epoxygenase in mediating BK channel–dependent K secretion in the CCD. Previous studies demonstrated that an DDMS increase in tubular fluid–flow rate stimulated net K C N P o secretion in the rabbit CCD and that the flow-in- duced increase in K secretion depended on BK 12 channel activity. Thus, net K (JK) and Na (JNa) MS-PPOH fluxes were measured in isolated rabbit CCDs C treated with or without MS-PPOH. We confirmed the previous observation that an increase in tubu-

lar fluid–flow rate enhanced both JNa absorption and K secretion.12 Data summarized in Figure 8A show that raising tubule flow rate from 1 to 5 Ϯ nl/mm per min increased JNa from 13.6 1.6 to Figure 4. (A) A channel recording showing the effect of AA on BK channels in 77.3 Ϯ 6.5 pmol/mm per min (n ϭ 3; P Ͻ 0.05) and the CCD in the presence of 5 ␮M indomethacin (top), DDMS (middle), or 10 J from Ϫ9.7 Ϯ 0.6 to Ϫ21.5 Ϯ 1.4 pmol/mm per ␮ K M MS-PPOH (bottom). The experiments were performed in a cell-attached min (n ϭ 3; P Ͻ 0.05). Inhibiting CYP-epoxygen- patch in the CCD from rats on an HK diet. The holding potential was 0 mV, and ␮ ␮ ase with 10 M MS-PPOH had no significant effect the channel closed level is indicated by C. (B) The effect of 10 MAAonBK Ϯ ␮ ␮ on both JNa (19.1 4.8 pmol/mm per min) and JK channels in the presence of 5 M indomethacin (Indo), 5 M DDMS, and 10 Ϯ ␮M MS-PPOH. *Significantly different from the corresponding control value. (9.9 1.3 pmol/mm per min) at the slow tubular flow rate; however, application of MS-PPOH com- Ϫ Ϯ that application of 100 nM 11,12-EET mimicked the effect of pletely abolished the effect of high flow rate on JK ( 11.1 1.4 AA on BK channels. Moreover, in the presence of 100 nM pmol/mm per min). In contrast, inhibiting CYP-epoxygenase 11,12-EET, adding AA failed to increase BK channel activity did not affect the effect of high flow rate on Na absorption, further, suggesting that the effect of AA and 11,12-EET is not which increased from 19.1 Ϯ 4.8 to 73.7 Ϯ 5.6 pmol/mm per additive (data not shown). To examine whether the effect of min (n ϭ 3; P Ͻ 0.05). The notion that CYP-epoxygenase plays 11,12-EET on BK channels was specific, we also tested the ef- a key role in mediating BK channel–dependent K secretion in

516 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 513–523, 2009 www.jasn.org BASIC RESEARCH

C toxin (IBX), an agent that specifically inhibits BK channels and has been previously shown to abol- ish flow-stimulated K secretion in rabbit CCD.12 Figure 8B summarizes the results from four such experiments showing that luminal application of

50 nM IBX had no effect on JK in the presence of Ϫ Ϯ MS-PPOH (control JK 26.7 3.8 pmol/mm per Ϫ Ϯ C min; MS-PPOH 9.5 0.5 pmol/mm per min; IBXϩMS-PPOH Ϫ11.4 Ϯ 1.2 pmol/mm per min). In contrast, neither IBX nor MS-PPOH had a significant effect on J (Figure 8B, right). These C Na data strongly indicate that CYP-epoxygenase is involved in flow-stimulated and BK channel–de- pendent K secretion in the CCD.

DISCUSSION

Figure 5. A channel recording showing the effect of 100 nM 11,12-EET on BK The kidney plays a key role in maintaining plasma channels in the CCD from rats on an HK diet. The experiments were performed K level in a normal range through secretion of K in a cell-attached patch, and the holding potential was 0 mV. The channel closed to match the dietary intake.1 Renal K secretion level is indicated by C and a dotted line. The top trace shows the time course takes place in the late distal tubule, CT, and the of the experiment, and two parts of the recording indicated by numbers and a CCD by a two-step mechanism1,3: K enters the bar are extended to show the fast time resolution. cell through Na-K-ATPase at basolateral mem- brane and leaves the cell through apical K chan- A C ϩ nels. Two types of K channels, a Ca2 -activated BK C and a ROMK-like SK channel, are expressed in the apical membrane of the CCD and are responsible for K secretion.3–6,8,31 It is generally accepted that C ROMK-like SK channels are mainly responsible for K secretion under normal dietary K intake10; however, C N P o when the tubule flow rate is high or dietary K intake increases,12,13,32 both BK channels and ROMK-like SK are involved in mediating K secretion. Because BK channels have a low open probability B and small numbers in the apical membrane under C basal conditions, BK channels must increase the channel open probability or numbers to secrete K in the CCD when dietary K intake increases. In this C study, we demonstrated that AA activates BK chan- nels in the CCD. Two lines of evidence indicate that the stimulatory effect of AA on BK channels is not the result of a nonspecific lipid effect: (1) The stimulatory Figure 6. A channel recording showing the effect of 100 nM 8,9-EET and effect of AA could not be mimicked by 11,14,17-EA; 11,12-EET (A) and 14,15-EET (B) on BK channels in the CCD from rats on an and (2) the effect of AA on BK channels was abolished HK diet. The experiments were performed in a cell-attached patch, and the in the presence of MS-PPOH, an inhibitor of CYP- holding potential was 0 mV. The channel closed level is indicated by C and epoxygenase. Thus, AA specifically stimulates the BK a dotted line. (C) The effect of 100 nM 8,9-, 14,15-, and 11,12-EET on BK channels in the CCD and could play a role in medi- channels in the CCD. Experiments were performed in cell-attached patches. ating K secretion in response to HK intake. *Significant difference (n ϭ 5 to 8) between the experimental and corre- The HK intake–induced stimulation of renal K se- sponding control values. cretion is mediated by both an aldosterone-depen- dent and an aldosterone-independent mechanism. response to high tubule flow rate was further tested by exam- HK intake stimulates the aldosterone secretion, which aug- 33,34 ining the effect of MS-PPOH on JNa and JK in CCDs perfused at ments the activity of both Na-K-ATPase and ENaC. This a flow rate of 5 nl/mm per min in the presence 50 nM iberio- leads to increasing the driving force for K secretion across the

J Am Soc Nephrol 20: 513–523, 2009 EET Activates BK Channels in the CCD 517 BASIC RESEARCH www.jasn.org

response to HK intake. The main products of CYP-␻-hydrox- C ylase are hydroxyeicosatetraenoic acids (HETE) such as 19- or 20-HETE.28 Although 20-HETE has been shown to inhibit a variety of K channels, including the apical 70 pS K channels in the thick ascending limb37 and BK channels in vascular smooth 38,39 C muscle cells, 20-HETE has been reported to activate BK channels in smooth muscles in airway40; however, the finding that application of 10 ␮M AA could still activate BK channels in the presence of DDMS, an inhibitor of CYP-␻-hydroxylase, ␻ C excluded that CYP- -hydroxylase–dependent AA metabolites are responsible for the stimulatory effect of AA on BK chan- nels. Thus, our results strongly indicate that CYP-epoxygen- ase–dependent AA metabolic pathway is responsible for the C AA-induced stimulation of BK channels in the CCD. We previously demonstrated that high dosages of AA also inhibited ROMK channels in both native CCD tubules and oocytes injected with ROMK channels.41 Because both ROMK and BK channels are involved in mediating K secretion in the CCD, it is paradoxic that AA has two opposite effects on both Figure 7. A channel recording showing the effect of AA (10 ␮M) apical K secretory channels; however, this discrepancy may be and 11,12-EET (100 nM) on BK channels in the CCD treated with MS-PPOH. The experiments were performed in a cell-attached due to different experimental conditions: The inhibitory effect patch in the CCD from rats on an HK diet, and the holding of AA on ROMK channels was observed only in inside-out potential was 0 mV. The channel closed level is indicated by C patches, whereas the stimulatory effect of AA on BK channels and a dotted line. The top trace shows the time course of the was observed in intact cells. We speculate that the inhibitory experiment, and three parts of the recording indicated by num- effect of AA on ROMK channels could not be demonstrated in bers and a bar are extended to show the fast time resolution. cell-attached patches because AA either would be metabolized by enzymes such as CYP-epoxygenase or was not in free form apical membrane. Moreover, HK could stimulate renal K se- in intact cells of the CCD under physiologic conditions. cretion by an aldosterone-independent mechanism. Mi- The major CYP-epoxygenases expressed in the rat CCD are croperfusion studies revealed that HK intake stimulated K se- CYP2J2 and CYP2C23.21,22 Although we could not carry out cretion in isolated perfusion CCDs from adrenalectomized Western blot to examine the expression of CYP2C23 and rabbits30; however, the signaling pathway underlying the aldo- CYP2J2 in the CCD, previous studies demonstrated that sterone-independent mechanism by which HK intake stimu- CYP2C23 was highly expressed in the thick ascending limb and lates renal K secretion and apical K channel activity has not distal nephrons, including CCD.22 Also, immunostaining been identified. Two lines of evidence suggest that CYP-epoxy- showed that CYP2J2 was located in the collecting duct and in genase–dependent AA metabolism may be involved in the reg- the proximal tubules.21 Thus, we speculate that HK intake ulation of renal K secretion in response to HK intake. First, HK should increase the expression of CYP2C23 in the CCD. This intake stimulates the expression of CYP2C23 and increases the notion is also supported by the finding that HK intake in- 11,12-EET concentrations in the CCD; second, CYP-epoxyge- creased 11,12-EET levels in the isolated CCD. Because HK in- nase–dependent AA metabolites stimulated the BK channels, take increased only the expression of CYP2C23 but not CYP2J2 which are known to mediate K secretion in response to in- in the renal cortex and outer medulla, CYP2C23 may play an creased K intake.13 important role in the regulation of K secretion in response to In addition to CYP-epoxygenase, COX and CYP-␻-hy- HK intake. The effect of HK on CYP2C23 expression could not droxylase have been shown to be able to metabolize AA in the be the result of stimulating aldosterone concentration because renal tubules, including CCD.28 The COX-dependent AA me- low Na intake, a maneuver that also stimulates aldosterone

tabolites such as E2 have been shown to activate secretion, inhibits the expression of CYP2C23 and 11,12-EET BK channels in cultured rabbit CCD by increasing intracellular production in the CCD.22 Thus, the effect of HK intake on ϩ Ca2 release35; however, the observation that inhibition of CYP2C23 expression is through an aldosterone-independent COX did not abolish the stimulatory effect of AA on BK chan- mechanism. The mechanism by which HK stimulates nels excluded the possibility that the COX-dependent metab- CYP2C23 expression is not clear but may be related to a reduc- olites of AA were responsible for the stimulatory effect of AA tion in the degradation or increase in the transcription of ep- on BK channels. Moreover, we previously demonstrated that oxygenase. Further experiments are required to explore these HK intake suppressed the expression of COX-2 expression in possibilities. the kidney.36 Thus, it is unlikely that COX-dependent AA me- Because it is extremely difficult to patch the rabbit tabolites are responsible for stimulating BK channel activity in CCD,6,42,43 we did not carry out the patch-clamp experiments

518 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 513–523, 2009 www.jasn.org BASIC RESEARCH

A

5 nl/min.mm * MS-PPOH 5 nl/min.mm 1 nl/min.mm 1 nl/min.mm MS-PPOH

5 nl/min.mm * 5 nl/min.mm * No MS-PPOH 1 nl/min.mm 1 nl/min.mm No MS-PPOH

0 20406080100 0 -5 -10 -15 -20 -25

JNa (pmol/mm.min) JK (pmol/mm.min)

B

IBX+ IBX+ MS-PPOH MS-PPOH *

MS-PPOH MS-PPOH *

Control Control

0 20406080100 JNa (pmol/mm.min) 0 -8 -16 -24 -32

JK (pmol/mm.min) ␮ Figure 8. (A) Effect of tubular fluid–flow rate on JNa and JK in the presence or absence of 10 M MS-PPOH. The rabbit CCD was treated with MS-PPOH (n ϭ 3) or vehicle (n ϭ 3) for 10 min before experiments, and 10 ␮M MS-PPOH was present throughout the entire ␮ ϩ experiments. (B) Effect of MS-PPOH (10 M) and luminal IBX (50 nM) MS-PPOH on JNa (left) and JK (right) in CCDs perfused at 5 nl/mm per min. Control measurements were determined in the absence of the inhibitors. *Significant difference (P Ͻ 0.05).

to examine whether AA could stimulate BK channels via the vating BK channels.15,16 The mechanism by which 11,12-EET CYP-epoxygenase–dependent pathway in rabbit CCD. Thus, stimulates BK channels in the CCD is not clear. Because 11,12- we used IBX as a tool to study the role of BK channels in me- EET has also been demonstrated to activate L-type Ca2ϩ chan- diating MS-PPOH–sensitive K secretion in response to in- nels50 and transient receptor potential channels,51 it is possible creased luminal flow rate. The observation that application of that 11,12-EET may stimulate BK channels by increasing influx IBX failed to cause further inhibition of K secretion in CCDs of external Ca2ϩ into the cell. BK channels have been observed perfused at a fast flow rate and treated with MS-PPOH strongly in both intercalated cells (ICs) and PCs, although channel ac- implicates EET in mediating BK channel–dependent and flow- tivity in IC exceeds that detected in PC52; however, the BK stimulated K secretion in the CCD. We speculate that 11,12- channel activity observed in the split-open CCD may be differ- EET is responsible for activating BK channels in the rabbit ent from that in the native tubule in vivo because these chan- CCD because 11,12-EET is the major product of EET in rabbit nels are normally closed in the absence of membrane stretch, ϩ kidney.44 Rabbit kidney expresses CYP2C family epoxygen- depolarization, and/or a high intracellular Ca2 concentra- ase,45 and purified rabbit CYP2C1 and 2C2 epoxygenase are tion. The observation that 11,12-EET stimulates BK channels able to metabolize AA and generate 11,12-EET (48%) and in PCs suggests that BK channels in PCs are involved in flow- 14,15-EET (16%).44 Moreover, EETs were isolated and puri- stimulated K secretion; however, we could not exclude the role fied from rabbit kidney,46 suggesting a possible role of EET in of BK channels in ICs in K secretion in the CCD. Moreover, BK the regulation of renal function. channels in ICs may play an important role in K recycling CYP-epoxygenase is able to convert AA to four EETs: 5,6- across the apical membrane, thereby regulating K absorption EET, 8,9-EET, 11,12-EET, and 14,15-EET.18,47 EETs have been through K/H-ATPase in response to dietary K intake and met- reported to exhibit diverse biologic activities, including dila- abolic need. tion of preglomerular microvessels,48 stimulation of angiogen- We previously demonstrated that 11,12-EET inhibited esis,49 and inhibition of sodium transport.20 It is reported that ENaC in the CCD. In this study, we showed that 11,12-EET 8,9- and 14,15-EET could also regulate BK channels23,24; how- activated BK channels in the CCD. The opposite effect of ever, the observation that only 11,12-EET stimulates BK chan- 11,12-EET on ENaC and BK channels has a significant physi- nels strongly suggests that 11,12-EET mediates the effect of AA ologic importance. HK intake is known to increase circulating on BK channels in the CCD. 11,12-EET has also been proposed level of aldosterone, which activates ENaC activity; however, to act as an endothelial-derived hyperpolarizing factor by acti- HK also increases 11,12-EET production, which inhibits ENaC

J Am Soc Nephrol 20: 513–523, 2009 EET Activates BK Channels in the CCD 519 BASIC RESEARCH www.jasn.org activity and suppresses Na absorption in the CT and CCD. channels and mediating BK channel–dependent K secretion in Because inhibiting ENaC should lead to reduction of the driv- the CCD. ing force for K secretion, increasing apical K channel activity should increase the apical K permeability and offset the effect of decreasing the electrical gradient on K secretion. Moreover, CONCISE METHODS the observation that inhibiting CYP-epoxygenase only in- creased the basal level of Na absorption but did not augment Preparation of CCDs the high flow rate–induced stimulation of JNa suggests that We used pathogen-free Sprague-Dawley rats of either gender (5 to 6 EET does not play a role in modulating flow-induced stimula- wk) in the experiments (Taconic Farms, Inc., Germantown, NY). Rats tion of Na transport. Alternatively, an increase in tubular flow were maintained on an NK diet (1%) or HK (10%) diet for 7 d. Rats rate could directly stimulate ENaC activity possibly by altering (Ͻ90 g) were killed by cervical dislocation, and kidneys were imme- the cleaved versus noncleaved ENaC at the apical membrane diately removed. The kidney was cut into several 1-mm slices from regardless the presence of 11,12-EET.53 Because HK intake has which the CCDs were isolated. The isolated CCD was placed on a 5 ϫ also been shown to increase the tubule flow rate in the distal 5-mm coverglass coated with polylysine, and the coverglass was trans- nephron,54 the high flow rate could directly activate ENaC and ferred to a chamber (1000 ␮l) mounted on an inverted Nikon micro- thereby increase the driving force for K secretion. Increased scope. The CCDs were superfused with HEPES-buffered NaCl solu- tubule flow rate has been shown to stimulate BK channel– tion. The protocol was reviewed and approved by an independent dependent K secretion. The mechanism by which high flow animal use committee from New York Medical College. For gaining rate stimulates BK channel activity is not completely under- access of the apical membrane, the CCD was cut open with a sharp- stood. It has been shown that increased tubule flow rate caused ened micropipette. For measurement of 11,12-EET levels in the CCD, ϩ ϩ the Ca2 influx12,55 and raised the intracellular Ca2 concen- kidneys were perfused with 0.5% collagenase-containing ringer, and trations, which should stimulate Ca2ϩ-dependent BK chan- kidney slices were incubated with the 0.5% collagenase-containing nels; however, the flow-induced increase in intracellular Ca2ϩ Ringer for an additional 3 to 5 min before dissection. is moderate and may not be sufficient to activate fully BK chan- nels alone, although the magnitude of the flow-stimulated in- Patch-Clamp Technique crease in intracellular Ca2ϩ in the immediate vicinity of BK An Axon200A patch-clamp amplifier was used to record channel cur- channels has not been explored.55 We speculate that increased rent, which was low pass–filtered at 500 Hz by an eight-pole Bessel flow rate raises the intracellular Ca2ϩ, which could lead to filter (902LPF; Frequency Devices, Haverhill, MA). The K current was stimulate Ca2ϩ-dependent phospholipase A2 activity. As a recorded and digitized by an Axon interface (Digidata 1300). Data consequence, AA release is enhanced and is converted to 11,12- were analyzed using the pClamp software system 9.0 (Axon, Sunny- EET, which stimulates BK channels and K secretion in the vale, CA). Channel activity defined as NPo was calculated from data CCD. In this regard, it is likely that EET levels measured in samples of 60-s duration in the steady state as follows: isolated and not perfused CCDs may not represent the true NP ϭ⌺(t ϩ 2t ϩ ...it) (1) EET level in vivo, and they provide only a relative index for the o 1 2 i

CYP-epoxygenase activity in the CCD from animals on differ- where ti was the fractional open time spent at each of the observed ent K diet. Consequently, EET level in split-open tubule may current levels. The PCs were recognized by their typical hexagonal not be high enough to reach the threshold for a full activation shape and large flat surface and confirmed by the presence of ROMK of BK channels. Thus, it is possible the BK channel activity in and ENaC in the apical membrane. All patch-clamp experiments were the split-open tubule is underestimated even in animals on an carried out in PCs and in cell-attached configuration in this study. HK diet. Also, we selected patches in which no ROMK channel activity was The view that CYP-epoxygenase could play a role in flow- detected for studying the effect of AA on BK channels. This selection stimulated net K secretion is strongly suggested by the finding allows us to calculate BK channel activity accurately in each patch. that inhibition of CYP-epoxygenase abolished the effect of high flow rate on K secretion in the CCD. Because HK intake Measurement of EET also increases tubule flow rate,54 it is possible that HK-induced The isolated CCDs were placed in a tube containing ice-cold Na increase in tubule flow is partially responsible for high 11,12- Ringer (0.5 ml). in the tubule and media were acidified to

EET levels in the CCD from HK-adapted rats. Moreover, it is pH 4.0 with 9% formic acid. After addition of 2 ng of D8 11,12-EET in conceivable that inhibiting CYP-epoxygenase could also atten- the tube as internal standard, the samples were extracted twice with uate BK channel–dependent K secretion in response to HK 2ϫ Vol ethyl acetate. Ethyl acetate extract was evaporated to dryness, intake; however, it is likely that factors other than EET are also and the lipid residue was subsequently resuspended in methanol. Af- involved in stimulating BK channel–dependent K secretion in ter extraction, the CCD tubules were homogenized and the protein response to HK intake. We conclude that HK intake stimulates concentration was measured. The samples were purified by reverse- ␮ ϫ the expression of CYP2C23 in the kidney and 11,12-EET levels phase HPLC on a C18 Bondapak column (4.6 24.0 mm) using a in the CCD and that the CYP-epoxygenase–dependent AA linear gradient from acetonitrile:water:acetic acid (62.5:37.5:0.05%) metabolic pathway plays an important role in activation of BK to acetonitrile (100%) over 20 min at a flow rate of 1 ml/min. The

520 Journal of the American Society of Nephrology J Am Soc Nephrol 20: 513–523, 2009 www.jasn.org BASIC RESEARCH

fraction containing 11,12-EET was collected on the basis of the elu- epithelial osmotic gradients, and thus water transport was assumed to tion profile of standards monitored by ultraviolet absorbance (205 be zero. Three to four samples of tubular fluid were collected under nm). The fractions were evaporated to dryness and resuspended in water-saturated light mineral oil by timed filling of a calibrated 30-nl 100 ␮l of acetonitrile. HPLC fractions containing 11,12-EET were volumetric constriction pipette at each perfusion rate (approximately derivatized as described previously.56 The derivatized 11,12-EET was 1 and 5 nl/mm per min). To determine the concentrations of K and dried with nitrogen and resuspended in 50 ␮l of iso-octane for gas Na delivered to the tubular lumen, we added ouabain (200 ␮M) to the chromatography–mass spectrometry analyses. A 1-␮l aliquot of deri- bath at the conclusion of each experiment to inhibit all active trans- vatized CYP-derived AA metabolites, dissolved in iso-octane, was in- port, and we obtained an additional three to four samples of tubular jected into a GC (Hewlett Packard 5890; Hewlett Packard, Boise, ID) fluid for analysis. The cation concentrations of perfusate and collected column. We used temperature programs ranging from 150 to 300°C tubular fluid were determined by helium glow photometry, and the at rates of 25°C/min, respectively.57 Methane was used as a reagent gas rates of net transport (in pmol/mm per min tubular length) were at a flow resulting in a source pressure of 1.3 torr and the, mass spec- calculated using standard flux equations, as described previously.58 trometer (Hewlett-Packard 5989A) was operated in electron capture The calculated ion fluxes were averaged to obtain a single mean rate of chemical ionization mode. The endogenous 11,12-EET (ion m/z 319) ion transport for the CCD at each flow rate. The flow rate was varied was identified by comparison of gas chromatography retention times by adjusting the height of the perfusate reservoir. The sequence of

with authentic D8 11,12-EET (m/z 327) standards. flow rates was randomized within each group of tubules to minimize any bias induced by time-dependent changes in ion transport. As Preparation of Renal Tissue for Western Blot indicated, some tubules were pretreated with MS-PPOH (10 ␮M) or The renal cortex and the outer medulla were separated and suspended IBX (50 nM) for 10 min before and then throughout the period of in RIPA buffer solution (1:8 ratio, wt/vol) containing 1ϫ PBS, 1% time that tubular fluid collections were made. Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS, and 10 ␮l of PMSF (10 mg/ml stock solution in isopropanol). A cocktail of protease inhibitors (10 ␮l; Sigma, St. Louis, MO) was added per mil- Statistic Analysis liliter of buffer at the time of lysis. The samples were homogenized on The bath solution for patch-clamp experiments contained (in mM)

ice for 15 min with a mortar and pestle, and the suspension was incu- 140 NaCl, 5 KCl, 1.8 CaCl2, 1.8 MgCl2, and 10 HEPES (pH 7.4). The ␮ bated at 4°C for1hinthepresence of DNAse (5 g/ml). After cen- pipette solution was composed of (in mM) 140 KCl, 1.8 MgCl2, and 5 trifugation, the supernatant was collected. We measured protein con- HEPES (pH 7.4). AA, 11,12-EET, DDMS, and MS-PPOH were pur-

centrations using a Bio-Rad Dc protein assay kit (Hercules, CA). chased from Cayman Chemical (Ann Arbor, MI). Indomethacin was Proteins homogenized from renal tissue were separated by elec- purchased from Sigma-Aldrich; 11,14,17-EA was obtained from Nu- trophoresis on 8 to 10% SDS–polyacrylamide gels and transferred to Check (Elysian, MN). The data are presented as means Ϯ SEM. We Immuno-Blot polyvinylidene difluoride membrane (Bio-Rad). The used t test to determine the statistical significance. P Ͻ 0.05 was con- membrane was blocked with Odyssey blocking buffer for fluorescence sidered to be significant. Western blotting (Rockland, Gilbertsville, PA) and incubated with the primary antibody at 4°C for 12 h. The membrane was washed four times (each 5 min) with PBS containing 0.1% Tween 20 followed by ACKNOWLEDGMENTS incubation with the secondary antibody for an additional 30 min. The membrane was then washed three times (10 min for each wash) with This study was supported by National Institutes of Health grants PBS and scanned by Odyssey infrared imaging system (LI-COR, Lin- HL34300 (W.-H.W.), DK38470 (L.M.S.), and P30 DK079307 coln, NE) at wavelength of 680 or 800 nM. (L.M.S., through the Pittsburgh Center for Kidney Research).

Microperfusion of Isolated Rabbit CCD We used adult (Ͼ6 wk) female New Zealand White rabbits (Covance, DISCLOSURES Denver, PA) housed in the Mount Sinai School of Medicine Center None. for Comparative Medicine. All animals were allowed free access to tap water and food. Animals were killed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory REFERENCES Animals. The animal protocol was approved by the Institutional An- imal Care and Use Committee at the Mount Sinai School of Medicine. 1. Giebisch G: Renal potassium transport: Mechanisms and regulation. Rabbit kidneys were removed via a midline incision, and single tu- Am J Physiol Renal Physiol 274: F817–F833, 1998 bules were dissected freehand in cold (4°C) Ringer’s solution contain- 2. Li DM, Wang ZJ, Sun P, Jin Y, Lin DH, Hebert SC, Giebisch G, Wang

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