INTRODUCTION

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HYPOKALEMIA HAS BEEN REPORTED to impair urinary concentrating ability in humans and animals (6, 22, 23) and to result in Cl wasting (27). Because the thick ascending limb (TAL) has a key role in the urinary concentrating ability, it has been proposed that a diminished Na transport in the TAL of the loop of Henle was involved in the hypokalemia-induced decrease in urinary concentrating ability (6). This hypothesis is supported by several independent studies. Gutsche et al. (12) have shown that Na transport in the TAL was inhibited in kidneys from hypokalemic rats. Similar results were also reported by Unwin et al. (29). Moreover, micropuncture studies have reported that K depletion reduced Cl reabsorption in the loop of Henle of rat kidneys (17, 18). Thus the data strongly indicate that an impaired NaCl transport in the medullary TAL (mTAL) is responsible for the diminished urinary concentrating ability induced by hypokalemia.

Recently, it has been demonstrated that the protein expression levels of Na-K-Cl cotransporters and ROMK, a renal K channel that is responsible for K recycling (2), were significantly lower in kidneys from rats on a K-deficient diet than those from animals on normal chow (20). Because K recycling is essential for the function of the cotransporter, a decrease in the apical K conductance is expected to inhibit the Na-K-Cl cotransporter. Thus studying the regulation of the apical K channels in the mTAL may elucidate the mechanism by which K depletion impairs the epithelial transport in the mTAL.

Eicosanoids have an important role in regulating the membrane transport in the mTAL. PGE₂ has been demonstrated to attenuate the effect of vasopressin on Cl reabsorption (5) and to inhibit Na reabsorption in the loop of Henle (26). It was recently reported that PGE₂ inhibits the reabsorption of bicarbonate in the mTAL (9). In addition, cytochrome P-450-dependent metabolites of arachidonic acid (AA) are involved in the regulation of transport function of the mTAL. Cytochrome P-450 metabolites of AA such as 20-hydroxyeicosatetraenoic acid (20-HETE) and 20-COOH-AA have been shown to inhibit the Na-K-Cl transporter (7) and bicarbonate reabsorption in the mTAL (10). We have reported previously that 20-HETE inhibited the apical 70-pS K channel (34). However, the role of eicosanoids in mediating the effect of K intake on transport function in the TAL is not clear. Because the 70-pS K channels make a significant contribution to the apical K conductance, we explored the role of 20-HETE in mediating the effect of dietary K intake on the apical K channels in the mTAL.

	METHODS

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Preparation of mTAL. Pathogen-free Sprague-Dawley rats (Taconic Farms, Germantown, NY) were used in the experiments. Animals were kept either on a high-K diet (10%, wt/wt) or on a K-deficient diet for 7-10 days before use. The method for preparation of the mTAL has been described previously (34). To immobilize the mTALs, we placed the tubules on a 5 × 5-mm cover glass coated with Cell-Tak (Collaborative Research, Bedford, MA). The cover glass was transferred to a chamber mounted on an inverted microscope (Nikon, Melville, NY), and the tubules were superfused with bath solution containing (in mM) 140 NaCl, 5 KCl, 1.8 MgCl₂, 1.8 CaCl₂, 5 glucose, and 10 HEPES (pH 7.4). We used a sharpened pipette to open the mTAL to gain access to the apical membranes.

Measurement of 20-HETE. Freshly isolated mTAL tubules (100-200 µg protein) were resuspended in 100-µl bath solution and incubated for 10 min at 37°C. The incubation was terminated by decreasing temperature to 0°C with ice. As an internal standard, 1 ng of deuterated [14,15-²H₂]20-HETE was mixed with the tubules. The tissue was spun down, and the supernatant was collected and evaporated. Distilled water (1 ml) was added to the tube containing the tubules, and lipids were extracted with ethyl acetate acidified with formic acid (pH 3.5). The extract was dissolved in 100 µl methanol and separated by reverse-phase HPLC using a gradient of acetonitrile in water (50-100% in 20 min) at a flow rate of 1 ml/min. The 20-HETE fraction was dried, resuspended in 100 µl acetonitrile, and converted to pentaflurobenzyl (PFB) ester by adding 10 µl PFB bromide and 10 µl of N,N-diisopropylethylamine. The mixture was incubated at room temperature for 30 min. The sample was evaporated under nitrogen and further incubated with 80 µl N,O-bis(trimethylsilyl)trifluoroacetamide for 30 min to collect the PFB ester, trimethylsilyl (TMS) ether derivative of 20-HETE. The sample was dried and dissolved in 50 µl of isooctane for gas chromatography-mass spectrometry (GC-MS) analysis (HP 5989A mass spectrometer interfaced with a HP 5890 gas chromatograph). The samples were injected into a 10 × 0.25-mm DB-1 capillary column with 0.25-µm film thickness (J & W Scientific, Folsom, CA). Helium was used as the carrier gas to raise the temperature to 180-300°C with 25°C/min of step increase. A selected ion monitoring negative chemical ionization (NCl) was used to record ion abundances at mass-to-charge ratio (m/z) 391 and m/z 393, which corresponded to the endogenous and deuterated derivatized 20-HETE (PFB ester TMS ether), respectively. The concentration of total 20-HETE in the purified biological samples was calculated by comparison of the ion abundance ratio (m/z 391/393) vs. a standard curve of 20-HETE-PFB-TMS/²H₂-20-HETE-PFB-TMS molar ratio constructed by NCl GC-MS analysis.

Patch-clamp technique. Electrodes were pulled with a Narishige model PP83 vertical pipette puller and had resistances of 4-6 M when filled with 140 mM NaCl. The channel current recorded by an Axon 200A patch-clamp amplifier was low-pass filtered at 1 kHz using an eight-pole Bessel filter (902LPF; Frequency Devices, Haverhill, MA). The current was digitized at a sampling rate of 44 kHz using a VR-10B digital data recorder (Instrutech) and stored on videotape (Hitachi FX600). For analysis, data stored on the tape were transferred to an IBM-compatible Pentium computer (Gateway 2000) at a rate of 4 kHz and analyzed using the pClamp software system v. 6.04 (Axon Instruments, Burlingame, CA). Channel activity was defined as NP_o, a product of channel open probability (P_o) and channel number (N). The NP_o was calculated from data samples of 30- to 60-s duration in the steady state as follows
where t_i is the fractional open time spent at each of the observed current levels. Because three types of K channels have been identified in the mTAL (1, 28, 31), we measured the channel current at three different membrane potentials in each patch to estimate the conductance of the K channel in the patch.

Solution and statistics. The pipette solution was composed of (in mM) 140 KCl, 1.8 Mg₂Cl, 1.8 Ca₂Cl and 5 HEPES (pH 7.4). Staurosporine, calphostin C, and 17-octadecynoic acid (17-ODYA) were purchased from Biomol and dissolved in pure ethanol. Indomethacin and nordihydroguaiaretic acid were obtained from Sigma and dissolved in methanol. The final concentrations of ethanol or methanol were less than 0.1% and had no effect on channel activity. Data are presented as means ± SE. We used paired and unpaired Student's t-tests to determine the statistical significance.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We examined the activity of apical K channels, as defined by NP_o, in the mTAL obtained from animals on a K-deficient diet and on a high-K diet for 7-10 days, and results are summarized in Fig. 1_. In the mTAL from rats on a K-deficient diet, the mean NP_o/patch of the 70- and 30-pS K channels were 0.11 ± 0.01 and 0.18 ± 0.01 (n = 41), respectively. In tubules from rats on a high-K diet, the mean NP_o/patch of the 70- and 30-pS K channel increased to 0.80 ± 0.07 and 0.60 ± 0.06 (n = 63), respectively. Thus it is apparent that the activity of the apical K channels was significantly lower in the mTAL from rats on a K-deficient diet than those on a high-K diet. Because previous studies have indicated that 20-HETE blocked the 70-pS K channel in the mTAL (34), we examined the possibility that an increase in 20-HETE production was responsible for decreasing the activity of the apical 70-pS K channel in the mTAL from rats on a K-deficient diet. We used GC-MS to measure the intracellular 20-HETE concentrations. Figure 2 is a representative recording from four such experiments showing that the 20-HETE concentration in the mTAL from rats on a K-deficient diet was 4.6 pg/µg protein. This was significantly higher than that observed in tubules from rats on a high-K diet (0.8 pg/µg protein).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Channel activity, defined by the number of channels in a patch and the open probability of single channels (NPo)/patch, of the apical 30- and 70-pS K channels in the tubules from rats on a high-K diet or on a K-deficient diet (low K), respectively. *Difference is significant (P < 0.05).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   20-Hydroxyeicosatetraenoic acid (20-HETE) concentrations measured by gas chromatography-mass spectrometry in the tubules from animals on a K-deficient diet (left) and on a high-K (middle) diet, respectively. Bottom trace shows the peak of 20-HETE standard. Right: results of 4 experiments. *Significantly different from control, P < 0.05.

If an increase in 20-HETE concentrations was responsible for decreasing the channel activity, inhibition of cytochrome P-450 -hydroxylation of AA with agents such as N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) (21, 30) should increase channel activity in the mTAL from rats on a K-deficient diet. We examined the effect of DDMS on the 70-pS K channel (30) in the tubules obtained from rats on a K-deficient or a high-K diet. Figure 3 shows the effect of DDMS on the apical 70-pS K channel in the mTAL from rats on a K-deficient diet. In the absence of DDMS, channel activity was low (NP_o = 0.05) in this particular case. Addition of 5 µM DDMS stimulated the activity of the 70-pS K channel and increased NP_o to 1.90. Figure 4 summarizes the results of experiments in which the effect of DDMS on channel activity was tested. Inhibition of cytochrome P-450 oxygenase increased the mean NP_o of the 70-pS K channel from 0.11 ± 0.01 to 0.91 ± 0.1 (n = 17) in the tubules from rats on a K-deficient diet. In contrast, DDMS had no significant effect on the activity of the 30-pS K channel (Fig. 4). This suggested that high concentrations of cytochrome P-450 metabolites of AA were involved in suppressing the activity of the 70-pS K channel in the tubules from rats on a K-deficient diet. In contrast, inhibiting cytochrome P-450 metabolism of AA caused a modest increase in NP_o of the 70-pS K channel in the mTAL from rats on a high-K diet (Fig. 5). In 19 experiments, we observed that inhibiting cytochrome P-450 metabolism of AA increased NP_o by 25 ± 3%, from 0.64 ± 0.07 to 0.81 ± 0.1, in the tubules harvested from rats on a high-K diet.

View larger version (43K): [in this window] [in a new window]   Fig. 3.   Channel recording showing the effect of N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) on the activity of the 70-pS K channel in the medullary thick ascending limb (mTAL) from animals on a K-deficient diet. The experiments were performed in a cell-attached patch, and the pipette holding potential was 0 mV. Top trace is time course of the experiments. Two parts of the data, indicated by numbers, are extended at a fast-time resolution. The channel closed level is indicated by C.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Channel activity (NPo/patch) of the apical 30- and 70-pS K channels in the mTAL from animals on a K-deficient diet. The channel activity was measured before and after treatment with DDMS/17-octadecynoic acid (17-ODYA) for 20 min. *Significantly different from control, P < 0.05.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Channel recording showing the effect of DDMS on the activity of the 70-pS K channel in the mTAL from animals on a high-K diet. The experiments were performed in a cell-attached patch, and the pipette holding potential was 0 mV. Top trace is time course of the experiments. Two parts of the data, indicated by numbers, are extended at a fast-time resolution. The channel-closed level is indicated by C.

It has been reported that renal PGE₂ production increased during hypokalemia (17, 18). To determine the role of cyclooxygenase metabolites of AA in mediating the effect of dietary K intake on channel activity, we explored the effect of indomethacin on channel activity. Figure 6 summarizes results demonstrating that indomethacin (5 µM) increased NP_o slightly from 0.12 ± 0.02 to 0.25 ± 0.06 (n = 6) in the mTAL from rats on a K-deficient diet. This suggests that cyclooxygenase metabolites of AA are not mainly responsible for suppressing channel activity in the mTAL from animals on a K-deficient diet. Also, Fig. 6 shows that inhibiting lipooxygenase with 5 µM nordihydroguaiaretic acid had no effect on channel activity (NP_o = 0.11 ± 0.02; n = 3).

View larger version (10K): [in this window] [in a new window]   Fig. 6.   Effects of indomethacin (5 µM) and nordihydroguaiaretic acid (NDGA, 5 µM) on 70-pS K channel activity in the mTAL from rats on a K-deficient diet. Experiments were performed in cell-attached patches. *Significantly different from control, P < 0.05.

To exclude the possibility that the sensitivity of the 70-pS K channel to 20-HETE diminished in the mTALs from rats on a high-K diet, we examined the effect of 20-HETE on the 70-pS K channel in an inside-out patch in the mTAL from animals on a high-K diet or on a K-deficient diet. Figure 7 shows that the dissociation constant value required for inhibition of the channel activity by 50% is ~5 nM in the tubules obtained from animals on a high-K diet as well as on a K-deficient diet. This finding excluded the possibility that the diminished response of the 70-pS K channel to inhibition of P-450 metabolism of AA is due to an alteration in the sensitivity of the 70-pS K channel to 20-HETE. This notion was also supported by observations that application of 20-HETE (100 nM) inhibited channel activity in a cell-attached patch by 90% in the presence of DDMS and that removal of 20-HETE restored the channel activity (Fig. 8).

View larger version (12K): [in this window] [in a new window]   Fig. 7.   Dose-response curves of the 70-pS K channel to 20-HETE in the mTAL from rats on a high-K diet () and on a K-deficient diet (). The experiments were carried out in inside-out patches.

View larger version (32K): [in this window] [in a new window]   Fig. 8.   Effect of 20-HETE (100 nM) on the 70-pS K channel in the mTAL from a rat on a high-K diet. DDMS was present throughout the experiments that were carried out in a cell-attached patch. Top trace is time course of the experiments. Three parts of the data are extended to show the fast-time resolution. The channel closed level is indicated by C.

That high concentrations of 20-HETE were responsible for decreasing channel activity in the mTAL from rats on a K-deficient diet was also indicated by observations that pretreatment of the mTAL with DDMS significantly increased the activity of the 70-pS K channels (Fig. 9). The pretreatment of the tubules with DDMS/17-ODYA increased NP_o from 0.10 ± 0.01 to 0.76 ± 0.07 (n = 34). In contrast, pretreatment of the mTALs failed to increase the channel activity significantly in the mTALs from animals on a high-K diet (control 0.80 ± 0.07, experiment 0.87 ± 0.07; n = 37).

View larger version (14K): [in this window] [in a new window]   Fig. 9.   Channel activity (NPo/patch) of the apical 70-pS K channels in the mTAL from animals on a K-deficient diet (low K) and on a high-K diet. The channel activity was measured before and after treatment with DDMS/17-ODYA for 20 min. *Significantly different from control, P < 0.05.

	DISCUSSION

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The TAL is responsible for the reabsorption of 20-25% of the filtrated Na load and has a key role in urinary concentrating mechanisms (11, 13). Na enters the cell through the luminal Na-K-Cl cotransporter, energized by a favorable electrochemical gradient of Na, and then is pumped out of the cell through the basolateral Na-K-ATPase. K recycling is essential for maintaining the normal function of the Na-K-Cl cotransporter (11, 14). First, K recycling hyperpolarizes the cell membrane and accordingly provides the driving force for Cl leaving the cell. Second, K recycling participates in generating the lumen positive potential, which is the driving force for transepithelial Na, Ca²⁺, and Mg²⁺ transport. Third, K recycling is important for providing an adequate supply of K to the Na-K-Cl cotransporter in the cortical TAL where K concentration is at least one order of magnitude lower than that of Na and Cl.

Three types of K channels (1, 31), Ca²⁺-activated large-conductance (>100 pS), intermediate-conductance (70-74 pS), and small conductance (30-40 pS), have been identified in the apical membrane of the rat TAL (28). The large-conductance K channel has a low open probability under physiological conditions. Thus it is unlikely that the large-conductance K channel could play a significant role in K recycling across the apical membrane (32). It is generally believed that the 70- and 30-pS K channels are mainly responsible for K recycling (32) because both channels have a high channel open probability and are frequently found under physiological conditions (1, 31). Furthermore, patch-clamp experiments revealed that the 70- and the 30-pS K channel contribute to the apical K conductance by ~80 and 20%, respectively (34). Thus the 70-pS K channel should have a key role in K recycling.

In the present study we have found that the apical 30- and 70-pS K channels are regulated by a dietary intake of K; a high intake of K increases, whereas a low intake of K decreases the activity of the apical K channels. Because K recycling is essential for maintaining normal function of the Na-K-Cl cotransporter, it is conceivable that diminished K conductance in the apical membrane could reduce the turnover rate of the cotransporter. Thus it is possible that the K depletion-induced decrease in channel activity in the mTAL is involved in decreasing NaCl transport and urinary concentrating ability in the kidney. The mechanism by which the dietary K intake changes the apical K channel activity in the mTAL is not completely understood. It was reported that K-depletion decreases the expression of ROMK channels as well as the Na-K-Cl cotransporter (20). This indicates that a downregulation of the K channels and the cotransporters in the mTAL should be partially responsible for the hypokalemia-induced impairment of NaCl transport. On the other hand, the expression of both ROMK channels and the Na-K-Cl cotransporter is the same in the tubules from animals on a high-K diet as those on a normal diet (20). This indicates that a posttranslation is also involved in mediating the effect of the dietary K intake on the apical K channels and the cotransporters. This notion is strongly suggested by the present finding that inhibiting cytochrome P-450-dependent metabolism of AA increased the activity of the apical 70-pS K channel in the mTAL from rats on a K-deficient diet. Because we could observe the effect of DDMS in 15 min, it is unlikely that the effect of DDMS was the result of increasing protein synthesis of the 70-pS K channel. Therefore, it is most likely that inhibition of cytochrome P-450 metabolism of AA activates the previously silent K channels. This view is also supported by our unpublished observations that the NP_o of the 70-pS K channel increased after excision to form inside-out patches in the mTAL from rats on a K-deficient diet. This suggests that the channel activity is suppressed by an endogenous inhibitor. Because renal PGE₂ concentrations increased significantly during hypokalemia, PGE₂ was suggested to be responsible for impairing urinary concentrating ability (17, 18). However, addition of indomethacin to block cyclooxygenase could only partially improve the transport function of the TAL in the loop of Henle (17), suggesting that PGE₂ is not mainly responsible for the hypokalemia-induced impairment of NaCl transport in the TAL. This notion also has been supported by the finding that indomethacin only modestly increased channel activity in the mTAL from rats on a K-deficient diet.

Several lines of evidence indicate that 20-HETE, a major cytochrome P-450-dependent metabolite of AA in the mTAL (4), has a key role in mediating the effect of dietary intake of K on the apical 70-pS K channels. First, the production of 20-HETE was fourfold higher in the mTALs from animals on a K-deficient diet than from those on a high-K diet. Second, inhibition of cytochrome P-450 monooxidase resulted in a significantly larger increase in the activity of the 70-pS K channel in the mTAL from rats on a K-deficient diet than from those on a high-K diet. Third, pretreatment of the mTAL with DDMS/17-ODYA raised the NP_o of the 70-pS K channel in the mTAL from rats on a K-deficient diet to an extent similar to that observed in the tubules from animals on a high-K diet. We have demonstrated that the effect of inhibiting cytochrome P-450 monooxidase was significantly attenuated in the mTAL from rats on a high-K diet compared with those on a K-deficient diet. This was not the result of diminished response of the 70-pS K channel to 20-HETE because the dissociation constant value required for inhibition of the channel activity by 50% was almost identical in tubules from rats on a high-K diet or a K-deficient diet. Therefore, it is possible that an increase in 20-HETE production is involved in decreasing the activity of the apical 70-pS K channels.

The major isoform of cytochrome P-450 monooxidase in the mTAL is the CYP4A family (21, 30), which converts AA to 20-HETE and epoxides (epoxyeicosatrienoic acids, EETs) such as 11,12-EET and 5,6-EET (3, 19, 21). Several studies have demonstrated that EETs have an important role in the regulation of ion channels. Whereas application of 20-HETE inhibited the Ca²⁺-activated maxi-K channels, EETs have been reported to activate the maxi-K channel (15, 19). Our preceding observation that addition of 11,12-EET had no significant effect on the 70-pS K channel did not support the role of EETs in regulating the channel activity directly (16). Moreover, in the present study we have found that 5,6-EET had no effect on the activity of the 70-pS K channel (unpublished observation). However, we need further experiments to explore the possibility that EETs could reverse the inhibitory effect of 20-HETE. Accordingly, if concentrations of EETs fall in the mTAL from rats on a K-deficient diet, the inhibitory effect of 20-HETE should be enhanced.

Several studies have shown that 20-HETE is an important second messenger for regulation of renal function, including epithelial transport (7, 8, 24, 34). Escalante et al. (7, 8) have shown that 20-HETE inhibits the activity of Na-K-Cl cotransporter. We have demonstrated previously that 20-HETE mediated the effect of stimulation of Ca²⁺-sensing receptors and the effect of angiotensin II (16) on the apical 70-pS K channel (35). In addition, salt intake is an important regulator for 20-HETE production. An increase in Na intake has been reported to increase the formation of 20-HETE (19, 25). A decrease in 20-HETE generation in response to high intake of Na was involved in inducing the salt-sensitive hypertension in animal models (19). In the present study, we have observed that low intake of K could also increase the 20-HETE generation. However, the mechanism by which low intake of K enhanced the formation of 20-HETE is not clear. There are at least three possibilities: 1) the AA release may be stimulated in the mTALs from rats on a K-deficient diet, 2) the activity of cytochrome P-450 monooxidase may be upregulated, and 3) the ratio between 20-HETE and EETs may change in response to the dietary intake of K. Further experiments are required to explore the mechanism of the effect of dietary K intake on 20-HETE formation.

Another finding of the present study is that inhibition of cytochrome P-450 monooxidase had no significant effect on the 30-pS K channel. This observation is also consistent with our previous finding that 20-HETE failed to inhibit ROMK channels expressed in oocytes. On the other hand, it is clearly shown that the dietary intake of K regulates the number of the 30-pS K channel. Thus it is possible that the mechanisms by which the low-K intake decreases the activity of the apical 30 pS are different from those of the apical 70-pS K channel. We have shown previously that protein tyrosine kinase has a key role in mediating the effect of dietary K on the apical low-conductance K channels in the cortical collecting duct; inhibition of protein tyrosine kinase increased the number of the apical K channels (33). We have postulated that an increase in the activity of protein tyrosine kinase is responsible for suppressing the activity of the apical low-conductance K channels in the collecting duct from rats on a K-deficient diet. Because the apical 30-pS K channel in the mTAL and the apical low conductance (30- to 40-pS) K channel in the collecting duct are generally believed to be closely related to ROMK channels, it is possible that the same mechanism may be responsible for mediating the effect of dietary intake of K on both K channels.

We conclude that the activity of the apical K channels in the mTAL is regulated by dietary intake of K and that an increase in 20-HETE generation is involved in inhibiting the activity of the apical 70-pS K channels in the mTAL from rats on a K-deficient diet.