Low Na intake suppresses expression of CYP2C23 and arachidonic acid-induced inhi - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:32 |只看该作者 |倒序浏览 |打印

作者：Peng Sun, Dao-Hong Lin, Tong Wang, Elisa Babilonia, Zhijian Wang, Yan Jin, Rowena Kemp, Alberto Nasjletti, and Wen-Hui Wang作者单位：1 Department of Pharmacology, New York Medical College, Valhalla, New York; and 2 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut


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      【摘要】" Z  o1 ]4 Y8 B0 T/ c# r
   We previously demonstrated that arachidonic acid (AA) inhibits epithelial Na channels (ENaC) through the cytochrome P -450 (CYP) epoxygenase-dependent pathway ( 34 ). In the present study, we tested the hypothesis that low Na intake suppresses the expression of CYP2C23, which is mainly responsible for converting AA to epoxyeicosatrienoic acid (EET) in the kidney ( 11 ) and attenuates the AA-induced inhibition of ENaC. Immunostaining showed that CYP2C23 is expressed in the Tamm-Horsfall protein (THP)-positive and aquaporin 2 (AQP2)-positive tubules. This suggests that CYP2C23 is expressed in the thick ascending limb (TAL) and collecting duct (CD). Na restriction significantly suppressed the expression of CYP2C23 in the TAL and CD. Western blot also demonstrated that the expression of CYP2C23 in renal cortex and outer medulla diminished in rats on Na-deficient diet (Na-D) but increased in those on high-Na diet (4%). Moreover, the content of 11,12-epoxyeicosatrienoic acid (EET) decreased in the isolated cortical CD from rats on Na-D compared with those on a normal-Na diet (0.5%). Patch-clamp study showed that application of 15 µM AA inhibited the activity of ENaC by 77% in the CCD of rats on a Na-D for 3 days. However, the inhibitory effect of AA on ENaC was significantly attenuated in rats on Na-D for 14 days. Furthermore, inhibition of CYP epoxygenase with MS-PPOH increased the ENaC activity in the CCD of rats on a control Na diet. We also used microperfusion technique to examine the effect of MS-PPOH on Na transport in the distal nephron. Application of MS-PPOH significantly increased Na absorption in the distal nephron of control rats but had no significant effect on Na absorption in rats on Na-D for 14 days. We conclude that low Na intake downregulates the activity and expression of CYP2C23 and attenuates the inhibitory effect of AA on Na transport.- S. x  h: |" y/ g" X% ?& r6 b9 b
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11,12-epoxyeicosatrienoic acid; Na absorption; cortical collecting duct

THE KIDNEY PLAYS AN IMPORTANT role in maintaining the balance between Na intake and Na excretion and thus in the regulation of extracellular volume ( 31 ). Although only 10% filtered Na is reabsorbed in the distal nephron, the Na absorption in the distal nephron plays a key role in determining the final renal Na excretion and is also tightly regulated by hormones and dietary Na intake ( 31 ). Defective regulation of Na transport in the distal nephron has been shown to cause diseases such as Liddle syndrome and pseudohypoaldosteronism type II ( 2, 13, 29 ).
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The cortical collecting duct (CCD) is responsible for the hormone-regulated Na absorption which takes place by a two-step process: Na enters the cells across the apical membrane via epithelial Na channels (ENaC) ( 1, 7, 23 ) and is extruded from the cell across the basolateral membrane by Na-K-ATPase ( 12, 22 ). It is well established that the rate-limiting step for Na absorption in the CCD is the apical Na conductance ( 15 ), which is regulated by aldosterone and Na intake: a high aldosterone level or low Na intake augments ( 24 ), whereas an increased Na intake decreases apical ENaC channel activity ( 31 ).3 o2 f7 Z. q) ]9 U9 u3 R' I

The mechanism by which low Na or aldosterone increases ENaC activity has been extensively studied and a variety of signaling molecules has been identified to be responsible for mediating the effect of low Na on ENaC ( 3, 19, 30 ). We previously demonstrated that CYP epoxygenase-dependent metabolites such as 11,12-epoxyeicosatrienoic acid (EET) play an important role in the regulation of ENaC channel activity ( 34 ): inhibition of CYP epoxygenase increased, whereas stimulation of CYP epoxygenase-dependent metabolism decreased ENaC activity. Because the expression of CYP epoxygenase increased in response to a high Na intake ( 11 ), it is possible that increased 11,12-EET level is partially responsible for suppression of ENaC activity induced by high Na intake. Conversely, we speculate that epoxygenase-dependent AA metabolism may also play a role in mediating the effect of low Na intake on ENaC activity. This hypothesis is tested by the present study which shows that the inhibitory effect of AA on ENaC was suppressed by low Na intake.
      【关键词】 suppresses expression arachidonic acidinduced inhibition
   METHODS

Preparation of CCDs. We used pathogen-free Sprague-Dawley rats of either sex (5-6 wk) in the experiments (Taconic Farms, Germantown, NY). Rats were maintained on a normal diet (0.5% Na) or Na-deficient (Na-D) diet for 2-3 or 13-15 days. Rats were killed by cervical dislocation and kidneys were immediately removed. The kidney was cut into several 1-mm slices from which the CCDs were isolated. The isolated CCD was placed on a 5 x 5-mm cover glass coated with polylysine and the cover glass was transferred to a chamber (1,000 µl) mounted on an inverted Nikon microscope. The CCDs were superfused with HEPES-buffered NaCl solution and the temperature of the chamber was maintained at 37 ± 1°C by circulating warm water surrounding the chamber. The protocol was reviewed and approved by an independent Animal Use Committee from NYMC. To gain access of the apical membrane, the CCD was cut open with a sharpened micropipette. To measure 11,12-EET in the CCD, kidneys were perfused with 0.5% collagenase-containing Ringer and kidney slices were incubated with the 0.5% collagenase-containing Ringer for an additional 3-5 min before dissection.
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Patch-clamp technique. An Axon200A patch-clamp amplifier was used to record channel current which was low-pass filtered at 50 Hz by an eight-pole Bessel filter (902LPF, Frequency Devices, Haverhill, MA). The Na current was recorded and digitized by an Axon interface (Digidata 1200). Data were analyzed using the pClamp software system 7.0 (Axon). Channel activity defined as NP o was calculated from data samples of 60-s duration in the steady state as follows: ( Eq. 1 ), where t i was the fractional open time spent at each of the observed current levels. Because the software could only calculate fewer than six channel levels, we selected patches with fewer than six channels in our study. Also, we selected patches with similar basal channel activity to study the effect of AA or EET on ENaC. The PC cells were recognized by its typical hexagonal shape and large flat surface. This characterization has been used by other investigators ( 6, 23 ) and confirmed by the presence of ROMK and ENaC in the apical membrane.: s! E% K6 R; V6 a, t

Measurement of EET. The isolated CCDs were placed in a tube containing ice-cold Na Ringer (0.5 ml). Eicosanoids in the tubule and media were acidified to pH 4.0 with 9% formic acid. We added 2 ng D 8 11,12-EET in the tube as internal standard, the samples were extracted twice with 2 x vol ethyl acetate. Ethyl acetate extract was evaporated to dryness and the lipid residue was subsequently resuspended in methanol. After extraction, the CCD tubules were homogenized and the protein concentration was measured. The samples were purified by reverse phase (RP)-HPLC on a C 18 µBondapak column (4.6 x 24 mm) using a linear gradient from acetonitrile:water:acetic acid (62.5:37.5:0.05%) to acetonitrile (100%) over 20 min at a flow rate of 1 ml/min. The fraction containing 11,12-EET was collected on the basis of the elution profile of standards monitored by ultraviolet absorbance (205 nm). The fractions were evaporated to dryness and resuspended in 100 µl of acetonitrile. HPLC fractions containing 11,12-EET were derivatized as described earlier ( 4 ). The derivatized 11,12-EET was dried with nitrogen and resuspended in 50 µl of iso-octane for gas chromatography-mass spectrometry (GC-MS) analyses. A 1-µl aliquot of derivatized CYP-derived AA metabolites, dissolved in iso-octane, was injected into a GC (Hewlett Packard 5890) column (DB-1ms; 10.0 m, 0.25-mm inner diameter, 0.25-µm film thickness, Agilent). We used temperature programs ranging from 150-300°C at rates of 25°C/min, respectively ( 16 ). Methane was used as a reagent gas at a flow resulting in a source pressure of 1.3 Torr and the MS (Hewlett-Packard 5989A) was operated in electron capture chemical ionization mode. The endogenous 11,12-EET (ion m/z 319) was identified by comparison of GC retention times with authentic D 8 11,12-EET ( m / z 327) standards.% C3 \& ^3 k8 M; B# q
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Immunocytochemical staining. Kidneys were perfused with 50 ml PBS containing heparin (40 U/ml) followed by 200 ml of 4% paraformaldehyde. After perfusion, the kidneys were fixed with 4% paraformaldehyde for 12 h and dehydrated. We used Leica1900 cryostat (Leica) to cut kidney slices which were dried at 42°C for 1 h. The slides were washed with 1 x PBS for 15 min, and permeablized with 0.4% Triton dissolved in 1 x PBS buffer containing 1% BSA and 0.1% lysine (pH 7.4) for 15 min. Kidney slices were blocked with 2% goat serum for 1 h at room temperature and then incubated with antibodies to aquaporin 2 (AQP2; Alomone, Jerusalem, Israel), THP (ICN, Pharmaceutical, Aurora, OH), and CYP2C23 (a gift from Dr. J. H. Capdevila, Vanderbilt University) for 12 h at 4°C. Slides were thoroughly washed with 1 x PBS followed by incubation in second antibody mixtures in 0.4% Triton X-100 dissolved in 1 x PBS for 2 h at room temperature.' v0 \" D; F& u/ W* r- J: t7 q; L6 F

In situ microperfusion of distal tubule. Male Sprague-Dawley rats weighing 200 to 250 g were maintained either on a normal Na (0.5%) or on a Na-D for 2 wk until the day of the experiment. Three to five animals were used in each experimental group. The rats were anesthetized by intraperitoneal injection of 100 to 150 mg/kg body wt of Inactin (5-ethyl-5-( L -methylpropyl)-2-thiobarbituric acid) purchased from Sigma. Animals were kept on a thermostatically controlled surgical table to maintain body temperature at 37°C.
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The technique for microperfusion of distal tubules is similar to that described previously ( 8, 33 ). Briefly, a late distal tubule (from the late distal convoluted tubule to the initial part of the CCD) was selected and perfused from the kidney surface at a rate of 12 nl/min. Distal tubule fluid was collected from the second or third segment on the kidney surface ahead of an oil block. After each collection, the pipette was withdrawn from the tubule into the oil covering the surface of the kidney and a small amount of oil was aspirated into the tip of the collection pipette to prevent evaporation of the sample. One collection was made in each perfused tubule and three to five collections were obtained in each kidney. The perfusion solutions were contained 20 µCi/ml of low-sodium [ 3 H]methoxy-inulin for measuring volume absorption, and 0.1% FD & C green dye for identification of the perfused loops. The composition of the perfusion solution was as follows (in mM): 115 NaCl, 25 NaHCO 3, 4 KCl, 1 CaCl 2, 5 Na-acetate, 2.5 Na 2 HPO 4, 0.5 NaH 2 PO 4. Solutions was bubbled at room temperature with 5% CO 2 -95% O 2 before use. The pH was adjusted to 7.4 with a small amount of NaOH or HCl. The rate of fluid absorption ( J v ) was analyzed by the [ 3 H]inulin concentrations in the perfusate and collected fluid. The concentrations of Na in the perfusion solution and tubular fluid samples were measured by ultramicro-atomic absorption spectrophotometry ( 8, 33 ) and compared with known standards. Rates of Na ( J Na ) absorption were expressed per millimeter of distal tubular length.

Solution and statistics. The bath solution for patch-clamp experiments contained (in mM) 140 NaCl, 5 KCl, 1.8 CaCl 2, 1.8 MgCl 2, and 10 HEPES (pH 7.4). The pipette solution was composed of (in mM) 140 NaCl, 5 KCl, 1.8 Mg 2 Cl, and 5 HEPES (pH 7.4). AA was obtained from Nu-Check (Elysian, MN) and 11,12-EET was purchased from Biomol. N -methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH), an inhibitor of CYP epoxygenase ( 32 ), was synthesized in Dr. Falck?s laboratory, Southwestern Medical Center at Dallas. The data are presented as means ± SE. We used Student?s t -tests to determine the statistical significance. If the P value was 3 E  ]% b/ s& _# p' F: z
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RESULTS
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High Na intake has been shown to stimulate CYP2C44 expression in the kidney and urinary EET excretion in mice and that defective regulation of CYP2C44 in response to high Na intake resulted in hypertension ( 20 ). To further explore the relationship between dietary Na intake and CYP epoxygenase activity, we examined the effect of Na restriction on the expression of CYP2C23, a rat homolog of mouse CYP2C44 ( 25 ). We expect that if high Na upregulates CYP epoxygenase, low Na intake should have an opposite effect on CYP2C23. Figure 1 is a typical immunostaining showing the expression of CYP2C23 in the renal cortex and outer medulla of rats on either a normal-Na diet (0.5%; Fig. 1 A ) or Na-D for 2 wk ( Fig. 1 B ). It is apparent that Na restriction significantly diminished the expression of CYP2C23 in the kidney. We next used AQP2 as a marker of CD and THP as a marker of the thick ascending limb (TAL) to determine whether CYP2C23 was expressed in both nephron segments. We confirmed the previous finding that CYP2C23 is expressed in the CCD ( Fig. 2 ) ( 34 ). In addition, the enzyme was also expressed in the OMCD. Figure 2 is a confocal image demonstrating the expression of CYP2C23 in AQP2-positive CCD ( top ) and OMCD ( bottom ). Furthermore, confocal images demonstrated that CYP2C23 was expressed in the cortical and medullary TAL ( Fig. 3 ). However, as expected, Na restriction suppressed the expression of CYP2C23 in the AQP2-positive CD ( Fig. 1 ) and in the TAL (data not shown). Also, the AQP2 expression decreased in the kidney from rats on low-Na diet.
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Fig. 1. Immunostaining of CYP2C23 and aquaporin-2 (AQP2) in the renal cortex and outer medulla in the kidney from rats on a control Na diet (0.5%) and from rats on a Na-deficient for 14 days. The samples were placed in the same slide and treated in the same way. The primary and secondary antibody were diluted in 1:300 and 1:1,000, respectively.1 \/ n3 f5 h7 q  U/ M" Z0 r$ F
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Fig. 2. Confocal image showing that CYP2C23 is expressed in the cortical collecting duct (CCD) and outer medullary collecting duct (OMCD), which are marked by a positive AQP2 staining. The sample was obtained from rats on a normal-Na diet.

Fig. 3. Confocal image demonstrating that CYP2C23 is expressed in the cortical and outer medullary thick ascending limb (TAL) from rats on a normal-Na diet. TAL tubules are indicated by positive Tamm-Horsfall protein (THP) immunostaining.

The finding that Na restriction decreased the expression of CYP2C23 was also supported by Western blotting in which the effect of Na intake on the total protein expression of CYP2C23 was examined. Figure 4 A is a Western blot demonstrating that the expression of CYP2C23 was significantly diminished in the kidney from rats on Na-D for 14 days (30 ± 5% of the control value, P 9 {1 w& B9 ]% A* ?" N
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Fig. 4. A : Western blot showing the effect of Na intake on the CYP2C23 expression. B : summarizes data showing the effect of low Na intake on 11,12-EET levels in the isolated CCDs. C : Western blot demonstrating the expression of CYP2C23 in the renal cortex and outer medulla in rats on Na-deficient diet (Na-D) for 3 and 14 days. The homogenates from renal cortex and outer medulla (100 µg) were used for the Western blot, and the primary and secondary antibody was diluted in 1:1,000 and 1:2,000, respectively.2 U' L  i# ?/ B8 A1 L

Since 11,12-EET mediates the inhibitory effect of AA on ENaC ( 34 ), a decrease in CYP2C23 expression in the CCD is expected to attenuate the inhibitory effect of AA on ENaC. We used the patch-clamp technique to examine the effect of AA on ENaC in the CCD of rats on a Na-D for 2-3 days or for 13-15 days. The reason for selecting rats on Na-D for 2-3 days rather than using animals on a normal-Na diet is that it is technically difficult to identify ENaC in the CCD from rats on a normal-Na diet. Western blot has also confirmed that the expression of CYP2C23 in renal cortex and outer medulla was significantly higher in rats on Na-D for 3 days (75 ± 8% control value, n = 4) than those on Na-D for 14 days (31 ± 5%; Fig. 4 C ). Thus it is still justified to use the rats on 3-day Na-D as control to examine the role of CYP epoxygenase in the regulation of ENaC. Figure 5 A is a representative channel recording showing that application of 15 µM AA inhibited ENaC and reduced NP o from 1.9 to 0.4 in the CCD of the rats on Na-D for 3 days. In contrast, application of the same amount of AA modestly inhibited ENaC and decreased NP o from 1.6 to 1.2 in the CCD from rats on Na-D for 2 wk ( Fig. 5 B ). Data summarized in Fig. 6 show that the dose response of the AA effect on ENaC in the CCD from rats on a Na-D for 3 and 14 days, respectively. Application of 15 µM AA decreased channel activity by 77 ± 8% ( n = 6, P

Fig. 5. Channel recording demonstrating the effect of 15 M AA on the activity of ENaC in the CCD from rats on a Na-D for 3 days ( A ) or for 14 days ( B ). Channel closed level is indicated by C and a dotted line. The pipette solution contains 140 mM NaCl and the holding potential was 40 mV (equivalent a -40 mV herpolarization) for 3-day Na-D trace and 20 mV for 14-day Na-D trace, respectively.

Fig. 6. Bar graph shows the dose response of the AA effect on ENaC in the CCD from rats on a Na-D for 3 days and for 2 wk. *Difference of the AA effect between 3 and 14 days is significant ( P
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Fig. 7. Channel recording demonstrating that 100 nM 11,12-EET inhibits ENaC in the CCD from rats on Na-D for 3 days ( top 2 traces) or 14 days ( bottom 2 traces). The effect of 11,12-EET was observed within 5 min and the holding potential was 30 mV.. p6 I% M# \: [" @) o
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Fig. 8. Recording shows the effect of MS-PPOH (10 µM) on ENaC activity in the CCD of rats on a control Na diet. Top 2 traces demonstrate the channel activity under control conditions (without MS-PPOH). Bottom 2 traces are the channel activity after inhibiting expoxygenase. The channel closed level is indicated by C. The experiment was performed in a cell-attached patch and the holding potential 30 mV. The effect of MS-PPOH occurs within 10 min.* D/ p9 p" k8 d6 u5 z3 N" l
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After showing that CYP epoxygenase-dependent AA metabolism plays a role in the regulation of ENaC activity, we used a microperfusion technique to examine the effect of inhibiting CYP epoxygenase on Na transport in the distal nephron. It is expected that inhibition of CYP epoxygenase should increase Na absorption in the distal nephron from rats on a normal-Na diet but have a minimal effect in rats on Na-D. A late distal tubule (from the late portion of the distal convoluted tubule to the initial part of the CCD) was selected and perfused from the kidney surface for the study. Data summarized in Fig. 9 show that inhibition of epoxygenase with MS-PPOH (10 µM) significantly increased Na absorption in the distal nephron from 200 ± 28 to 322 ± 27 pmol·min -1 ·mm -1 ( n = 5). The stimulatory effect of MS-PPOH must be related to CYP epoxygenase activity because MS-PPOH had no significant effect on Na absorption in the rats fed on Na-D for 14 days. Figure 8 shows that Na transport before MS-PPOH was 211 ± 31 pmol·min -1 ·mm -1 in the distal nephron of K-restricted animal. This value was not significantly different from that observed in rats on a normal-Na diet. A similar finding has been reported by other investigators ( 28 ) and it is speculated that the Na delivery rate to the distal nephron normally used in microperfusion study was not high enough to demonstrate that low Na intake alters Na absorption. Thus a high perfusion rate may be required to saturate the capacity of Na absorption in the distal nephron. Although the Na absorption rate in the distal nephron between normal and Na-restricted rats was the same under our experimental conditions, inhibition of CYP epoxygenase with MS-PPOH failed to stimulate Na absorption and it was 221 ± 16 pmol·min -1 ·mm -1 in rats on Na-D for 14 days.

Fig. 9. Effect of inhibiting CYP epoxygenase with MS-PPOH (10 µM) on net Na absorption ( J Na ) of late distal tubule in rats on a normal-Na diet or a Na-D for 2 wk. *Significantly different from the control value ( P

DISCUSSION" C9 c. h! U  K# q' H! n; ~' p' N

The aim of the present study was to explore the role of CYP epoxygenase-dependent AA metabolism in mediating the effect of Na intake on ENaC activity and Na transport in the distal nephron. We selected the Na-restricted rats rather than those on a high-Na diet in the patch-clamp experiments because ENaC activity was suppressed in the CCD from rats on a high-Na diet. Thus it is difficult to study the effect of AA on ENaC in rats on a high Na intake or even on a normal-Na diet. As an alternative, we carried out the study in Na-restricted rats for 3 or 14 days to prove the principal that Na intake regulates the CYP epoxygenase-dependent AA metabolism which in turn affects ENaC activity and Na transport in the distal nephron. Three lines of evidence suggest that Na intake regulates the activity of CYP epoxygenase in the distal nephron: 1 ) Western blot shows that high Na increased while low Na decreased CYP2C23 expression; 2 ) the immunostaining of CYP2C23 was almost absent in the CD from rats on Na-D for 14 days; and 3 ) the level of 11,12-EET in the CCD of rats on a Na-D diet was significantly lower than those on a control Na diet. This is consistent with previous findings that high Na intake increases CYP epoxygenase-dependent AA metabolites such as 11,12-EET ( 17 ). Although we did not examine the expression of CYP epoxygenases other than CYP2C23 which are also expressed in the kidney ( 25 ), CYP2C23 is a main CYP epoxygenase responsible for converting AA to 11,12-EET in the kidney ( 11 ). This notion is also supported by the finding that a diminished expression of CYP2C44, a mouse homolog to rat CYP2C23, decreases urinary EET excretion in CYP4A10(-/-) mice ( 20 ). In addition to CYP epoxygenase, AA can also be metabolized by CYP hydroxylation in the kidney. We and others previously demonstrated that 20-HETE, a CYP hydroxylase-dependent AA metabolite, inhibited Na transport in the TAL by blocking apical K channels and Na-Cl-K cotransporter ( 5, 10 ). However, it is unlikely that the CYP-hydroxylase-dependent AA metabolism plays a significant role in mediating the effect of AA on ENaC because inhibition of CYP hydroxylase failed to block the effect of AA on ENaC ( 34 ).
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Several studies have suggested that CYP epoxygenase regulates renal Na transport. It has been reported that 5,6-EET inhibits Na transport in the rabbit CCD ( 26 ). The effect of 5,6-EET on Na transport is possibly mediated by a cyclooxygenase-dependent pathway because the effect of 5,6-EET was abolished by indomethacin. We have previously shown that adenosine inhibits ENaC activity by a CYP epoxygenase-dependent pathway ( 35 ). It is possible that stimulation of A 1 adenosine receptor increases AA release which is converted to 11,12-EET and blocks ENaC ( 34 ). The view that AA-induced inhibition of ENaC depends on CYP epoxygenase activity is supported by two lines of evidence: 1 ) The inhibitory effect of AA was attenuated from rats on Na-D for 2 wk which had a low CYP epoxygenase activity and 2 ) inhibition of epoxygenase increased ENaC activity in the CCD of rats on a normal-Na diet. Because 11,12-EET inhibits ENaC in the CCD with the same potency in rats on Na-D for 14 days as that for 3 days, this indicates that the attenuated AA effect on ENaC was due to a diminished CYP-epoxygenase activity. We speculate that the sensitivity of ENaC to AA in the CCD from rats on control (0.5%) Na diet and on a high Na intake should be much higher than that in rats on Na-D.& v# i: v  Y, Y$ Z6 ?

The notion that CYP epoxygenase AA metabolism plays a role in the regulation of Na transport in the distal nephron under physiological conditions was further indicated by the microperfusion study in which inhibition of CYP epoxygenase increased Na transport in the distal nephron in rats on a control Na diet. Because distal nephron includes connecting tubule and the initial portion of CCD where ENaC is located, it is assumed that inhibition of CYP epoxygenase should decrease 11,12-EET levels and thus increase Na transport by stimulation of ENaC activity. Since Na restriction downregulates the expression of CYP epoxygenase activity in the distal nephron, inhibition of CYP epoxygenase had no effect on ENaC in rats on a Na-D. Thus our data provide further evidence to support the role of CYP epoxygenase, especially CYP2C23/44, in the regulation of renal Na transport.- D1 F& K5 N! }0 Y- I
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The role of CYP epoxygenase-derived EETs in the regulation of Na transport was best demonstrated in CYP4A10(-/-) mice in which deletion of the CYP4A10 gene also suppressed the expression of CYP2C44, a major enzyme which converts AA to EETs in mice ( 20 ). As a consequence, the mice developed hypertension even if they were fed a normal-Na diet (0.5%). Three lines of evidence indicate that the hypertension in CYP4A10(-/-) mice was the result of defective regulation of ENaC by CYP2C44 (mouse homolog of CYP2C23)-dependent AA metabolism. First, Na intake failed to regulate the expression of CYP2C44 and renal 11,12-EET levels. Second, application of AA failed to inhibit ENaC in the CCD from CYP4A10(-/-) mice while 11,12-EET was able to inhibit ENaC. Third, treatment of animals with amiloride restored the normal blood pressure in CYP4A10(-/-) mice. Therefore, CYP2C44/23 is involved in mediating the effect of Na intake on renal Na transport.
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The mechanism by which Na intake regulates the expression of CYP2C23 is not clear. Because high Na suppresses while low Na increases the plasma level of aldosterone, it is possible that the renin-angiotensin-aldosterone pathway may play an important role in the regulation of CYP epoxygenase activity. The stimulatory effect of aldosterone or low Na intake on ENaC takes place not only at the level of transcription and translation but also at the posttranslational level by increasing the channel open probability ( 14 ) or the ENaC number in the apical membrane ( 21 ). However, the mechanism by which aldosterone increases the channel open probability is not completely understood. Aldosterone has been shown to increase SGK activity which increases the surface number and channel open probability of ENaC ( 18, 19 ). It has also been demonstrated that aldosterone stimulates methylation of ENaC and increased channel open probability of ENaC ( 27 ). The present observation that low Na intake diminished the inhibitory effect of AA on ENaC suggests that decreases in the 11,12-EET level induced by low Na intake are, at least in part, responsible for the aldosterone-mediated increase in channel open probability. This notion is also supported by the finding that channel open probability in the MS-PPOH-treated CCD is significantly higher than that without the CYP epoxygenase inhibitor ( 34 ). We speculate that the effect of aldosterone on ENaC activity is partially mediated by regulation of CYP epoxygenase and 11,12-EET levels in the CCD.8 U+ Z1 t- T0 m6 @

The present study has shown that CYP2C23 is expressed not only in the CCD but also in the TAL. However, the role of CYP2C23 in the TAL has not been explored. Because the expression of CYP2C23 in the TAL was also regulated by Na intake, this strongly suggests that CYP epoxygenase should also play a role in the regulation of Na transport in the TAL. Further experiments are needed to explore the role of EET in the regulation of Na transport in the TAL. In summary, the present study suggests that CYP epoxygenase-dependent AA metabolites play a role in mediating the effect of Na intake on Na transport in the distal nephron. High Na intake stimulates the expression of CYP epoxygenase and increases the formation of 11,12 EET which inhibits Na absorption. In contrast, Na restriction downregulates the expression of CYP epoxygenase and decreases the concentration of 11,12-EET. As a consequence, the channel open probability of ENaC increased. We concluded that low Na suppresses the expression of CYP2C23 in the CCD and decreases 11,12-EET levels which are partially responsible for increasing ENaC channel open probability in the CCD from Na-restricted rats.
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The work is supported by National Institutes of Health Grant HL-34300.

ACKNOWLEDGMENTS9 U6 C+ p* ~: J+ a+ L2 j9 L# T
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The authors thank M. Steinberg for editorial assistance in the preparation of the manuscript and Dr. J. Capdevila for providing the antibody of CYP2C23.
      【参考文献】, f1 L, ^& a7 ]1 c9 k4 b
Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, and Rossier BC. Amiloride-sensitive epithelial Na channel is made of three homologous subunits. Nature 367: 463-467, 1994.7 L& g# o. @  G. |/ J7 K

4 k+ E. A) N4 I9 C* X

Chang SS, Grunder S, Hanukoglu A, Rosler A, Mathew PM, Hanukoglu I, Schild L, Lu Y, Shimkets RA, Nelson-Williams C, Rossier BC, and Lifton RP. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet 12: 248-253, 1996.0 g8 d* ]8 q2 c8 k3 P5 B8 u# N

! Q/ W8 n+ d5 B: c7 F+ v$ c
Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sci USA 96: 2514-2519, 1999.

( t6 k1 `* I1 D' D- Y6 n  G

Croft KD, McGiff JC, Sanchez-Mendoza A, and Carroll MA. Angiotensin II release 20-HETE from rat renal microvessels. Am J Physiol Renal Physiol 279: F544-F551, 2000.# c# D# e, B$ k. |4 ~
; ^. f/ O( u* x; [) e& d% S
# `: o8 q8 z7 O4 A+ ^/ v5 O

Escalante B, Erlij D, Falck JR, and McGiff JC. Effect of cytochrome P450 arachidonate metabolites on ion transport in rabbit kidney loop of Henle. Science 251: 799-802, 1991.

0 a1 u7 _" n$ c8 A/ Q5 H

Frindt G and Palmer LG. Low-conductance K channels in apical membrane of rat cortical collecting tubule. Am J Physiol Renal Fluid Electrolyte Physiol 256: F143-F151, 1989.
& v4 Q( `2 _, C* [( {# t7 x
9 J; O8 C( T5 u& |) X

Garty H and Palmer LG. Epithelial sodium channel: Function, structure, and regulation. Physiol Rev 77: 359-396, 1997.7 z7 F3 N; A; Y. g" \/ A+ V
% V1 l' d( O9 \  L& ?0 ?' }
/ G8 o: r. E8 J* n& J& l
" |% R  J0 G) W, G2 E! p% C
Good DW and Wright FS. Luminal influences on potassium secretion: sodium concentration and fluid flow rate. Am J Physiol Renal Fluid Electrolyte Physiol 236: F192-F205, 1979.
; l5 a7 s1 _9 x
0 H" }: W0 {$ t% U

Gu RM, Wei Y, Jiang H, Balazy M, and Wang WH. The role of 20-HETE in mediating the effect of dietary K intake on the apical K channels in the mTAL. Am J Physiol Renal Physiol 280: F223-F230, 2001.3 b" n5 I) V1 A% C  `" g. v4 d* `

  s: S9 K: M6 Y1 ?

Holla VR, Makita K, Zaphiropoulos PG, and Capdevila JH. The kidney cytochrome P450 2C23 arachidonic acid epoxygenase is upregulated during dietary salt loading. J Clin Invest 104: 751-760, 1999.1 k5 _! u* u  y
  F% B/ `% N  P. P  Z5 Z
) t$ w; p8 L* V5 Z& W; f2 r, K
7 Y5 e$ y* {; U3 K3 ^" C+ F& G
Horisberger JD, Lemas V, Kraehenbuhl JP, and Rossier BC. Structure-function relationship of Na,K-ATPase. Annu Rev Physiol 53: 565-584, 1991.! G9 [" X1 k* K6 X6 R/ Q
8 V' ~, q+ V$ u5 ^
/ B2 U0 D3 t$ U6 l$ t

Kahle KT, Wilson FH, Leng Q, Lalioti MD, O?Connell AD, Dong K, Rapson AK, MacGregor GG, Giebisch G, Hebert SC, and Lifton RP. WNK4 regulates the balance between renal NaCl reabsorption and K secretion. Nat Genet 35: 372-376, 2003.

+ S# S' ~$ K5 F! ], D( K
Kemendy AE, Kleyman TR, and Eaton DC. Aldosterone alters the open probability of amiloride-blockable sodium channels in A6 epithelia. Am J Physiol Cell Physiol 263: C825-C837, 1992.

1 _0 n) K0 x+ i; E7 T
Koeppen BM and Stanton BA. Sodium chloride transport. In: The Kidney: Physiology and Pathophysiology, edited by Seldin DW and Giebisch G. New York: Raven, 1992, p. 2003-2039.) `0 g7 A" d' \: D  p' d3 A

1 q1 n: _/ a. R* z% ~

Macica CM, Balazy M, Falck JR, Mioskowski C, and Carroll MA. Characterization of cytochrome P450-dependent arachidonic acid metabolism in rabbit intestine. Am J Physiol Gastrointest Liver Physiol 265: G735-G741, 1993.
, L  |0 ^2 z# c+ n" l

Makita K, Takahashi K, Kerara A, Jacobson HR, Falck JR, and Capdevila JH. Experimental and/or genetically controlled alterations of the renal microsomal cytochrome P450 epoxygenase induce hypertension in rats fed a high salt diet. J Clin Invest 94: 2414-2420, 1994.
4 y1 _1 d+ c8 p& }2 i4 f, l- E

Muller OG, Parnova RG, Centeno G, Rossier BC, Firsov D, and Horisberger JD. Mineralocorticoid effects in the kidney: correlation between ENaC, GILZ, and Sgk-1 mRNA expression and urinary excretion of Na and K  . J Am Soc Nephrol 14: 1107-1115, 2003.) g  L6 w  ]% n; o  T6 r- _1 y) N' Z( n

% |9 ~0 h  O3 r* D
+ y9 [% y4 V# n9 B" D8 j$ p
Naaray-Fejes-Tóth A and Fejes-Tóth G. The sgk, an aldosterone-induced gene in mineralocorticoids target cells, regulates the epithelial sodium channel. Kidney Int 57: 1290-1294, 2000.+ C6 ~. Y) S6 y% k- F; {5 w

# t( {5 r) N; Y6 R1 g. `! \  v* \
Nakagawa K, Holla VR, Wei Y, Wang WH, Gatica A, Wei S, Mei S, Miller CM, Cha DR, Price EJ, Zent R, Pozzi A, Breyer MD, Guan Y, Falck JR, Waterman MR, and Capdevila JH. Salt sensitive hypertension is associated with a dysfunctional Cyp4a10 gene and kidney epithelial sodium channel. J Clin Invest. In press.& b5 x) O( i; D7 M# N8 P
; U! \9 @% t8 `: V  i6 C* D: s$ Q

. _* M! M9 {  V
Pácha J, Frindt G, Antonian L, Silver RB, and Palmer LG. Regulation of Na channels of the rat cortical collecting tubule by aldosterone. J Gen Physiol 102: 25-42, 1993.; h  @6 Y4 ^0 h/ w1 T. {% T
# Q3 X3 }7 O/ j# R( a, F

Palmer LG, Antonian L, and Frindt G. Regulation the the Na-K pump of the rat cortical collecting tubule by aldosterone. J Gen Physiol 102: 43-57, 1993.2 x2 i, u' j' `2 W7 j# F& B$ \

1 c- ^  J" e3 t+ j2 e! W
Palmer LG and Frindt G. Amiloride-sensitive Na channels from the apical membrane of the rat cortical collecting duct. Proc Natl Acad Sci USA 83: 2767-2770, 1986.- C0 e6 @* I8 e8 A* o: z1 f

# C9 {. {" p( T2 p3 d
Palmer LG and Frindt G. Regulation of apical membrane Na and K channels in rat renal collecting tubules by aldosterone. Semin Nephrol 12: 37-43, 1992.  o3 I$ s6 O# y0 u) V) |
" I5 ^8 D2 \* Q" g
0 N9 F! E2 I  N3 }4 w9 _2 R

Roman RJ. P450 metaboltes of arachidonic acid in the control of cardiovascular function. Physiol Rev 82: 131-185, 2004.
& p* k8 {5 T. O- y! M
. P) E. ]* N7 t& \- A3 c

Sakairi Y, Jacobson JR, Noland TD, Capdevila JH, Falck JR, and Breyer MD. 5,6-EET inhibits ion transport in collecting duct by stimulating endogenous prostaglandin synthesis. Am J Physiol Renal Fluid Electrolyte Physiol 268: F931-F939, 1995.9 g/ \( Q/ s; v3 i

1 Z4 u, ?) ]& Q7 Q, h- Q* s% Q

Sariban-Sohraby S, Burg M, Wiesmann WP, Chiang PK, and Johnson JP. Methylation increases sodium transport into A6 apical membranes vesicles: Possible mode of aldosterone action. Science 225: 745-746, 1984.

Schafer JA and Chen L. Low Na diet inhibits Na and water transport response to vasopressin in rat cortical collecting duct. Kidney Int 54: 180-187, 1998.+ C% ]4 V7 `* A$ g3 m+ B6 i! ]
% I; V4 Y3 D% O% O  h- y; z3 ?
; g) D/ W& |0 ?4 ~. d5 P

Shimkets RA, Warnock DG, Bositis CM, Nelson-Williams C, Hansson JH, Schambelan M, Gill JR Jr, Ulick S, Milora RV, Findling JW, Canessa CM, Rossier BC, and Lifton RP. Liddle?s syndrome: heritable human hypertension caused by mutations in the subunit of the epithelial sodium channel. Cell 79: 407-414, 1994.' O5 C; J0 _6 s+ K- [

7 e  x# T$ [1 X- Z

Stockand JD. New idea about aldosterone signaling in epithelia. Am J Physiol Renal Physiol 282: F559-F576, 2002.
- i8 H+ b5 k: k9 d4 |5 e8 n

" d% s/ |2 e- P' Z3 u+ C) r& G4 a" v7 a
Verrey F, Hummler E, Schild L, and Rossier BC. Control of Na transport by aldosterone. In: The Kidney: Physiology and Pathophysiology, edited by Seldin DW and Giebisch G. Philadelphia: Lippincott Williams & Wilkins, 2000, p. 1441-1472.2 w1 ~- h- L* b8 h/ `

7 Z! Z$ }9 G0 k- y2 N6 Z. ~! D
Wang MH, Brand-Schieber E, Zand BA, Nguyen X, Falck JR, Balu N, and Schwartzman ML. Cytochrome P450-derived arachidonic acid metabolism in the rat kidney:Characterization of selective inhibitors. J Pharmacol Exp Ther 284: 966-973, 1998.
7 }, s: ^9 [. E! v; s0 I: S! G8 B
# l5 _" T0 J+ v" l# `
; V5 Y/ k3 j# ~
Wang T and Giebisch G. Effects of angiotensin II on electrolyte transport in the early and late distal tubule in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 271: F143-F149, 1996./ V* T% c8 x" J0 m; \# w* p
# l" x; N6 B- O3 U( y' Q) ?

! R- S/ Z0 l1 [1 V
Wei Y, Lin DH, Kemp R, Yaddanapudi GSS, Nasjletti A, Falck JR, and Wang WH. Arachidonic acid inhibits epithelial Na channel via cytochrome P450 (CYP) epoxygenase-dependent metabolic pathways. J Gen Physiol 124: 719-727, 2004.

) t  ?/ P6 B6 `' z  m6 z
Wei Y, Sun P, Wang ZJ, Yang BF, and Wang WH. Adenosine inhibits ENaC by cytochrome P -450 epoxygenase-dependent metabolites of arachidonic acid. Am J Physiol Renal Physiol 290: F1163-F1168, 2006.

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