Distribution of cytochrome P-450 4A and 4Fisoforms along the nephron in mice - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:23 |只看该作者 |倒序浏览 |打印

作者：David E.Stec, AveriaFlasch, Richard J.Roman,  Jared A.White作者单位：1 Department of Physiology and Biophysics, Centerfor Excellence in Cardiovascular-Renal Research, University ofMississippi Medical Center, Jackson, Mississippi 39216-4505; and Department of Physiology, Medical College ofWisconsin, Milwaukee, Wisconsin 53226 4 A8 x: O' E$ {6 v8 e' _; s0 ?

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      【摘要】
   The production of20-hydroxyeicosatetraenoic acid (20-HETE) in the kidney is thought tobe involved in the control of renal vascular tone and tubular sodiumand chloride reabsorption. 20-HETE production in the kidney has beenextensively studied in rats and humans and occurs primarily via theactions of P -450 enzymes of the CYP4A and -4F families.Recent advancements in molecular genetics of the mouse have made itpossible to disrupt genes in a cell-type-specific fashion. Theseadvances could help in the creation of models that could distinguishbetween the vascular and tubular actions of 20-HETE. However, isoformsof the CYP4A and -4F families that may be responsible for theproduction of 20-HETE in the vascular and tubular segments in thekidney of the mouse are presently unknown. The goal of this study wasto identify the isoforms of the CYP4A and -4F families along the nephron by RT-PCR of RNA isolated from microdissected renal blood vessels and nephron segments from 16- to 24-wk-old male and female C57BL/6J mice. CYP4A and -4F isoforms were detected in every segment analyzed, with sex differences only observed in the proximal tubule andglomeruli. In the proximal tubular segments from male mice, the 4A10and -12 isoforms were present, whereas the 4A10 and -14 isoforms weredetected in segments from female mice. In glomeruli, sex differences inthe expression pattern of CYP4F isoforms were also observed, with malemice expressing the 4F13, -14, and -15 isoforms, whereas female miceexpressed the 4F13, -16, and -18 isoforms. These results demonstratethat isolated nephron and renal vessel segments express multipleisoforms of the CYP4A and -4F families; therefore, elimination of asingle CYP4A or -4F isoform may not decrease 20-HETE production in allnephron segments or the renal vasculature of male and female mice.However, the importance of CYP4A vs. -4F isoforms to the production of20-HETE in each of these renal tubular and vascular segments of themouse remains to be determined.
      【关键词】 cytochome P A isoforms cytochrome P F isoforms nephron segment mouse microdissection reverse transcriptionpolymerase chain reaction hydroxyeicosatetraenoic acid
   INTRODUCTION

20-HYDROXYEICOSATETRAENOIC ACID (20-HETE) is the majormetabolite of arachidonic acid (AA) produced in the kidney of several species, including humans ( 22 ). 20-HETE has been reportedto regulate renal vascular tone ( 12, 30 ), mediatetubuloglomerular feedback ( 31 ), and inhibit electrolytetransport in several nephron segments ( 6, 20 ). Alterationsin renal 20-HETE production may also contribute to the development ofhypertension in both the spontaneously hypertensive rat (SHR) andthe Dahl salt-sensitive (Dahl S) rat ( 21, 24, 25 ).
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Three isoforms of the CYP4A family have been cloned in the mouseand include the CYP4A10, CYP4A12, and CYP4A14 isoforms. Bell et al.( 2 ) identified CYP4A12 as the primary isoform that is constitutively expressed in the kidney of male and female mice, withlevels in males being much higher than those in females. The CYP4A10and CYP4A14 isoforms are expressed at lower levels in the mouse kidneyunder basal conditions; however, both isoforms were markedly inducibleby treatment with the peroxisome proliferator methylclofenapate( 2, 8 ). Although mouse CYP4A isoforms share a high degreeof amino acid identity with rat CYP4A isoforms ( 7 ), thecatalytic activity of mouse CYP4A10 and CYP4A12 isoforms in regard tothe production of 20-HETE has yet to be determined. In the humankidney, the only isoform of the CYP4A family to be cloned is CYP4A11( 2 ). However, in contrast to the rat, recent studies haveindicated that the primary isoforms responsible for the production of20-HETE in the human kidney are members of the CYP4F family ( 4, 16 ).8 l) |- l' S2 c9 Y
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In mice, five members of the CYP4F family have also been identified.The CYP4F15 and -16 isoforms have been demonstrated to be expressed inthe kidney under basal conditions, with the CYP4F16 isoform being themore abundant transcript ( 5 ). In the kidney, the CYP4F15isoform is highly inducible by clofibrate, whereas clofibrate has noeffect on CYP4F16 levels ( 5 ). Sex differences in thelevels of CYP4F expression in the kidney have yet to be reported.Although CYP4F isoforms in the human kidney have been demonstrated toproduce 20-HETE ( 4, 16 ), the ability of the CYP4F isoformsexpressed in the kidneys of mice and rats to metabolize AA and produce20-HETE has not yet been demonstrated.
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The dissection of complex paracrine systems has been successfullyaccomplished by either targeted overexpression or disruption ofspecific genes in transgenic and gene knockout mice. Advances in mousegenetics allow for targeted overexpression and deletion of genes inspecific nephron segments of the kidney ( 17, 26 ). Thedevelopment of these approaches would greatly increase our understanding of the physiological consequence of altered renal tubularor vascular 20-HETE production. Targeted disruption of the CYP4A14 gene has been described ( 10 );however, instead of decreasing renal 20-HETE production, loss of thisisoform was associated with increased production of 20-HETE in thekidney and hypertension in male mice. The increased 20-HETE production in these mice was attributed to an increase in the expression of theCYP4A12 isoform; however, changes in the expression of CYP4F isoformswere not considered in the previous study ( 10 ). Theresults from that study indicate that 20-HETE produced in the kidney ofthe mouse may be derived from multiple isoforms of the CYP4A and CYP4Ffamilies in a sex- and cell-type-specific fashion. In the presentstudy, we set out to identify the isoforms of the CYP4A and CYP4Ffamilies present in specific nephron segments and renal vessels of themouse. This was accomplished by designing specific primers to amplifythe individual isoforms of the CYP4A and CYP4F families and performingRT-PCR on bulk isolated nephron segments and renal microvessels fromboth male and female mice.
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METHODS% {. V1 P# Z, _  E) [) `

Animals. Studies were performed in 16- to 24-wk old male and female C57BL/6Jmice purchased from The Jackson Laboratory (Bar Harbor, ME). All micewere housed under standard conditions and allowed full access to foodand water. All procedures were approved by the Institutional AnimalCare and Use Committee at the University of Mississippi Medical Center.$ |, K9 j4 j( ~/ m- O, v( D  [8 Z7 J

Isolation of proximal tubules, thick ascending limb of the loopof Henle, and collecting ducts. Mice were anesthetized with pentobarbital sodium (50 mg/kg), and theabdominal aorta was cannulated below the kidneys. The mesenteric andceliac arteries were tied off to increase delivery of solutions to thekidneys. The kidneys were flushed through the aorta with 3 ml of coldwash solution consisting of (in mM) 135 NaCl, 3 KCl, 1.5 CaCl 2, 1 MgCl 2, 2 KH 2 PO 4, 5.5 glucose, 5 L -alanine,and 10 HEPES (pH 7.4). The kidneys were immediately removed, and thecortex and outer medulla were separated under a dissecting microscope.The cortex was then sectioned into 1-mm-thick coronal sections using aStadie-Riggs microtome, and the outer medulla was finely minced withscissors. Cortical sections as well as the outer medulla were thenincubated separately in dissection solution at 37°C for 1 hwhile O 2 was continuously blown over the mix. Thedissection solution consisted of wash solution with the followingadditives: 1 mg/ml collagenase (type II, 1.2 U/mg, Sigma, St. Louis,MO), 1 mg/ml hyaluronidase (359 U/mg, Sigma), and 1 mg/ml soybeantrypsin inhibitor (Sigma). Proximal tubules were then isolated byseparation on a Percoll gradient ( 28 ), and individualtubule segments (30 mm in total length) were collected under astereomicroscope and placed in 100 µl of lysis buffer forRT-PCR-specific RNA isolation (RNAqueous, Ambion, Austin, TX).0 J' a! [: T8 H

Thick ascending limbs of the loop of Henle (TALH) were isolated fromthe outer medulla of the kidney after enzymatic digestion, and thesupernatant was collected and placed on a 70-µm nylon sieve. Thesieve was rinsed several times with ice-cold digestion solution, andthe retained tissue was washed off the sieve with a wash solutioncontaining 1% BSA. Individual TALH segments were then collected undera stereomicroscope with fine forceps and placed into the lysis solutionfor RNA isolation. Collecting ducts were isolated from 1-mm-thickcoronal sections of the whole kidney after a 1-h incubation indigestion solution at 37°C while O 2 was continuouslyblown over the mix. After the digestion period, both cortical andmedullary collecting ducts were collected under a stereomicroscope withfine forceps and placed into the lysis solution for RNA isolation.
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Isolation of renal microvessels and glomeruli. Renal vessels were isolated from the kidney using a modified Evans bluetechnique ( 13 ). Briefly, the kidney was first flushed with3 ml of wash solution, followed by another 3 ml of wash solution containing 1% (wt/vol) Evans blue (Sigma). The kidneys were then hemisected and forced through a 180-µm nylon sieve. The remaining tissue was rinsed several times with wash solution containing 1% BSA.The tissue was then collected from the sieve and lightly homogenizedseveral times with a Polytron homogenizer. The resulting suspension wasthen passed several times first through a 20-gauge needle, and thenthrough a 22-gauge needle. The suspension was then passed though a180-µm mesh sieve that was atop a 100-µm mesh sieve. After severalwashes in wash solution with 1% BSA, the material retained by each ofthe sieves was collected. Each microvascular fraction was examinedunder a stereomicroscope, and most vessel segments were found to betotally devoid of glomeruli and tubules. Clean vessel segments werethen collected using fine forceps and placed into the lysis solutionfor RNA isolation. Glomeruli were isolated by a rapid sieving techniqueas previously described ( 23 ). The material passing throughthe 180-µm sieve after the kidney tissue was forced through wascollected and passed though a 100-µm sieve and then through a 70-µmsieve. The 70-µm sieve was rinsed several times in wash solution with1% BSA, and the material was collected. The fraction was then examinedunder a stereomicroscope, and individual glomeruli devoid of visible tubular contamination were collected (100 total) and place into lysissolution for RNA isolation.

RNA isolation and RT-PCR. RNA was isolated using a commercially available kit specificallydesigned for RT-PCR according to the manufacturer's guidelines (RNAqueous, Ambion). RNA was eluted in a 60-µl volume withnuclease-free water. Ten microliters of RNA were then used in a reversetranscription reaction using an oligo-dT primer with avianmyeloblastosis virus (AMV)-RT in a 20-µl final volume. Tocontrol for amplification of genomic DNA, reactions lacking AMV-RT wererun in parallel. PCR was performed on 1 µl of the reversetranscription reaction in a 25-µl volume using standard PCR reactionconditions. The PCR reactions for the CYP4A isoforms were cycled asfollows: 94°C for 30 s, 60°C for 1 min, and 72°C for 1 min.A total of 35 cycles was performed. The PCR conditions for the CYP4Fisoforms were identical, with the exception of a 55°C annealingtemperature. PCR primers for the CYP4A isoforms were as follows:CYP4A10 (GenBank accession no. AB018421 ), sense5'-GACAAGGACCTACGTGCTGAGG, antisense 5'-CTCATAGCAAATTGTTTCCCA; CYP4A12( Y10222 ), sense 5'-TGAGTCCTATGAAAGAGTGCC, antisense5'-CTGGAAGCCCAGCAGAAGGTG; and CYP4A14 ( Y11640 ), sense 5'-CCCACAGGGACATGCAGATTAG, antisense5'-CACACAGAGCTCGGAAGACC. The PCR primers for the CYP4A10,CYP4A12, and CYP4A14 amplified products of 473, 470, and 508 bp,respectively. PCR primers for the CYP4F isoforms were as follows:CYP4F13 ( AF233643 ), sense 5'-TGCATCCCCCAGTCTTATTA, antisenseCYP4F13- 5'-AGGAGGCAGTTCTGTTTATTCA; CYP4F14 ( AF233644 ), sense5'-AGCTCACCTCTGGCATTTATTCC, antisense 5'-CTCAGACATCCCTTTGGCTTCCTA;CYP4F15 ( AF233645 ), sense 5'-TCCGCTTTGACCCAGAGAATA, antisense5'-GTCAAGGCGATGGAAGTTTACC; CYP4F16 ( AF233646 ), sense 5'-GCCTGGCTGAGAAAAGTC, antisense 5'-TTATAAAAAGGAGGGGAAGC; and CYP4F18( AF233647 ), sense 5'-AAAGGTGTCATAAGCCGAATAAGT, antisense 5'-ACAGGTGGGTGGATGGATAGG. PCR primers for the CYP4F13, CYP4F14, CYP4F15, CYP4F16, and CYP4F18 amplified products of 479, 434, 542, 421, and 374 bp, respectively. As a positive control, GAPDH was amplified.The PCR primers for GAPDH were as follows: sense 5'-AAGAAGGTGGTGAAGCAGGCAT and antisense 5'-GATGGTATTCAAGAGAGTAGGGA. Theprimers amplified a 405-bp fragment. PCR products were separated on0.8% DNA-agar (Midwest Scientific, St. Louis, MO) gels. Experiments were repeated in four individual male and female mice. Gels were visualized under ultraviolet illumination, and images were captured using a gel documentation system with software supplied by the manufacturer (Gel Doc 2000, Bio-Rad, Hercules, CA). PCR products fromall isoforms amplified from either whole kidney or liver were clonedinto TA cloning vectors (Invitrogen, Carlsbad, CA). Two independentclones for each isoform were then sequenced to confirm specificity ofPCR primers.
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Measurement of AA metabolism. Mice were anesthetized with pentobarbital sodium (50 mg/kg), and thekidneys were flushed with an ice-cold washing solution as noted above.The kidneys were then rapidly removed and sectioned into cortex andouter medulla. Outer medullas from two mice were pooled to obtainsufficient amounts of tissue for the microsome preparation. Microsomesfrom the liver were also prepared. The tissue was homogenized in 3 mlpotassium phosphate buffer containing (in mM) 10 potassium phosphate,25 sucrose, 1 EDTA, and 0.1 PMSF. Microsomes were prepared bydifferential centrifugation and resuspended in a buffer that contained(in mM) 100 potassium phosphate, pH 7.2, 1 dithiothreitol, 1 EDTA, and0.1 PMSF as well as 30% glycerol.
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Enzyme activity was measured by incubating microsomes from the cortexand outer medulla of the kidney or the liver (0.5 mg protein) with asaturating concentration of [ 14 C]AA (1 µCi; 42 µM) inan NADPH-regenerating system as previously described ( 24 ).Additional experiments were then performed comparing the ability ofmicrosomes prepared from the kidneys of male and female rats tometabolize AA using a lower concentration of AA (1 µCi; 1.8 µM),because the percent conversion of AA to 20-HETE in renal microsomes ofC57/BL6 mice was found to be very low relative to rats, rabbits, andother species as reported previously ( 1 ). The reactionswere terminated by acidification with formic acid, extractions weremade twice with ethyl acetate, and the preparation was dried underN 2 gas. The metabolites were then resuspended in500 µl of 100% ethanol and separated by HPLC using a C 18 reverse-phase column (2.1 × 250 mm, 5 µm; Supelco) and a 2-cmguard column. A linear elution gradient ranging fromacetonitrile-water-acetic acid (50:50:2 vol/vol/vol) toacetonitrile-acetic acid (100:0.2 vol/vol) was used. Metabolites weremonitored by a radioactive flow detector modified with lead shieldingfor a low background. The mean production rate for each metabolite wascalculated and expressed as picomoles formed per minute per milligramof protein.
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Statistics. Values are presented as means ± SE. The significant difference inmean values was evaluated by an unpaired t -test. A value of P significant.5 O9 G- E5 ^% y$ F
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RESULTS( e* ^# t/ h% T8 \& J5 c% `8 L

Expression of CYP4A isoforms in nephron segments and renal vessels. In the proximal tubule, the CYP4A10 and CYP4A12 isoforms were the onlyisoforms detected in male mice (Fig. 1 A ). This was in contrast tofemale mice, in which the CYP4A10 and CYP4A14 isoforms were detected inthe proximal tubule (Fig. 1 B ). The CYP4A12 isoform could notbe amplified from proximal tubules isolated from female mice. TheCYP4A10 isoform was the only isoform that could be amplified from theTALH and collecting ducts of both male and female mice (Figs. 2 and 3 ).In the renal vessels, the CYP4A10 isoform was 180-µm diameter) and small(between 180- and 100-µm diameter) vessels (Fig. 4 ). There were no sex differences in theexpression pattern of CYP4A isoforms in the renal vasculature (Figs. 4 and 5 ). The CYP4A10 isoform was also theonly isoform expressed in isolated glomeruli from both male and femalemice (Fig. 6 ).2 S" V5 E4 Q& g8 F- X
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Fig. 1. RT-PCR distribution of CYP4A and CYP4F isoforms in isolatedproximal tubule segments of male ( A ) and female( B ) mice. The CYP4A isoforms detected in the male wereCYP4A10 and CYP4A12, whereas the CYP4A10 and CYP4A14 isoforms weredetected in females. The pattern of expression of the CYP4F isoformswas identical in males and females, with the CYP4F13, CYP4F14, CYP4F16,and CYP4F18 isoforms all being detected. M, 100-bp molecular ruler(Bio-Rad).
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Fig. 2. RT-PCR distribution of CYP4A and CYP4F isoforms in isolated thickascending loop of Henle (TALH) segments of male ( A ) andfemale ( B ) mice. Only the CYP4A10 isoform was detected inboth male and female mice. The CYP4F13 and CYP4F16 isoforms were bothdetected in isolated TALH segments from male and female mice.
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Fig. 3. Distribution of CYP4A and CYP4F isoforms in isolated cortical andmedullary collecting duct segments of male ( A ) and female( B ) mice by RT-PCR. The CYP4A10, CYP4F13, and CYP4F16isoforms were detected in both male and female mice.
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Fig. 4. 180-µm)renal vessels from male ( A ) and female ( B ) mice.The CYP4A10, CYP4F13, CYP4F16, and CYP4F18 isoforms were all expressedin large renal vessel segments from both male and female mice.  M. l7 I% G0 R8 E1 \

Fig. 5. RT-PCR distribution of CYP4A and CYP4F isoforms from small renalvessels from male ( A ) and female ( B ) mice.Vessels were 100 µm in diameter. The CYP4A10 andCYP4F13 isoforms were the only isoforms detected in both male andfemale mice.

Fig. 6. Distribution of CYP4A and CYP4F isoforms in isolated glomeruli frommale ( A ) and female ( B ) mice as determined byRT-PCR. The CYP4A10 isoform was detected in glomeruli from both maleand female mice. The CYP4F13, CYP4F14, and CYP4F15 isoforms were allpresent in glomeruli isolated from male mice. In glomeruli from femalemice, the CYP4F13, CYP4F16, and CYP4F18 isoforms were all detected.. r! e  K7 F% k3 D

Expression of CYP4F isoforms in nephron segments and renal vessels. Proximal tubule segments from both male and female mice exhibitedidentical patterns of expression of CYP4F isoforms. The CYP4F13,CYP4F14, CYP4F16, and CYP4F18 isoforms were all detected in proximaltubule segments from both sexes (Fig. 1, A and B ). In the TALH and collecting duct, the CYP4F13 and CYP4F16isoforms were detected in both male and female mice (Figs. 2 and 3 ). In the renal vasculature, the CYP4F13, CYP4F16, and CYP4F18 isoforms wereall detected in large vessels from both male and female mice (Fig. 4 ).However, the CYP4F13 isoform was the only isoform amplified from smallvessel segments in both sexes (Fig. 5 ). Sex differences in theexpression pattern of CYP4F isoforms were also apparent in glomeruli.In glomeruli from male mice, the CYP4F13, CYP4F14 and CYP4F15 isoformswere detected (Fig. 6 A ), whereas the CYP4F13, CYP4F16, andCYP4F18 isoforms were detected in glomeruli from female mice (Fig. 6 B ). The expression pattern of the CYP4A and CYP4F isoformsbetween male and female mice is summarized in Fig. 7.

Fig. 7. 180-µm diameter); SV,small renal vessels (between 180- and 100-µm diameter); Glom,glomeruli; PT, proximal tubule; TLH, thin decending and ascending loopsof Henle; TALH, thick ascending loop of Henle; CD, collectingducts.3 j* d0 Q! Z2 s+ r( [
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AA metabolism. We first characterized the renal metabolism of AA in the kidney andliver of male C57/BL6 mice using the same substrate concentration (42 µM) and incubation time (15-30 min) previously used in rats ( 1 ). The results of these experiments are presented inTable 1. Microsomes prepared from thelivers of these mice avidly produced 20-HETE; 14,15-, 11,12-, 8,9-, and5,6-diHETEs; 18-, 19-, 16- 15-, 12-, 5-, and other HETEs; and lesserquanities of epoxyeicosatrienoic acids (EETs). The overallproduction rate of 20-HETE was similar to the levels previouslyreported in rat liver microsomes ( 14 ). Mouse renalcortical microsomes produced primarily 20-HETE, with lesser quantitiesof EETs and diHETEs, and largely 15- and 12-HETEs. However, thecatalytic activity of the microsomes to produce 20-HETE and EETs wasabout five times lower than that seen in the livers of these same miceor comparable to the levels previously observed in renal corticalmicrosomes in rats ( 1, 14 ). We tried a number of differentsubstrate concentrations, cofactor additions, incubation times, andamounts of protein (0.25-2 mg) in the reactions but could notincrease conversion rates.

Table 1. Metabolism of arachidonic acid by microsomes prepared from liver andrenal cortex of male C57/BL6 mice

Because of the low conversion rates of AA to EETs and 20-HETE, wetherefore chose to compare the relative activity of microsomes preparedfrom the kidneys of male and female rats using only[ 14 C]AA (1.8 µM) to maximize the conversion rates andour ability to detect differences in activity. The production of20-HETE was lower in microsomes prepared from the renal cortices offemale mice compared with male mice (1.38 ± 0.13 vs. 1.71 ± 0.12 pmol · min 1 · mgprotein 1 ); however, this difference did not reachstatistical significance (Table 2 ). Theproduction of 20-HETE in the outer medulla of male and female mice wasnot statistically different. Epoxygenase activity was significantlylower in microsomes prepared from the cortex and outer medulla offemale mice compared with male mice, with the cortical values averaging2.90 ± 0.33 and 2.09 ± 0.24 pmol · min 1 · mg protein 1 in male and female mice, whereas outer medullary values averaged 1.49 ± 0.27 and 0.90 ± 0.53 pmol · min 1 · mg protein 1 (Table 2 ).
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Table 2. Sex differences in renal metabolism of arachidonic acid in C57BL/6Jmice  v+ A  J, }+ m; G# O# Z: G
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DISCUSSION5 b$ u3 v- Y4 Q* M8 z; `

The goal of the present study was to examine the distribution ofCYP4A and CYP4F isoforms along the nephron and in renal microvessels ofC57BL/6J mice to determine what 20-HETE-producing enzymes may bepresent in these tubular and vascular segments. This information isespecially useful in the context of defining an experimental approachto reduce the production of 20-HETE in one of these segments byelimination of a CYP4A or CYP4F isoform. Although the CYP4A10 andCYP4F13 isoforms were detected in every tubular and vascular segmentexamined, some segments also express other CYP4A and CYP4F isoformsthat may be able to produce 20-HETE if the CYP4A10 or CYP4F13 isoformswere inactivated by gene targeting. For example, in the TALH of micethe CYP4A10 isoform is present as well as two isoforms of the CYP4Ffamily. Elimination of this CYP4A isoform would leave the other CYP4Fisoforms still intact, which might be sufficient to produce 20-HETE inthis nephron segment. Previous studies have demonstrated that 20-HETEis a regulator of chloride transport in the TALH ( 6 ) andthat decreased production of 20-HETE in the TALH may be responsible forthe shifting of the pressure-naturetic response and the development ofhypertension in Dahl S rats ( 15 ). However, this hypothesismay be difficult to test in mice in which the CYP4A10 isoform has beendeleted in the TALH because this nephron segment expresses multipleCYP4F isoforms. Similarly, 20-HETE has been demonstrated to be a potent vasoconstrictor of the renal microcirculation ( 12 ), andalteration in the production of 20-HETE in renal arterioles is thoughtto contribute to the development of hypertension in the SHR ( 25, 29 ). Even though the CYP4A10 isoform was the only isoformdetected in renal blood vessels of male and female mice, disruption of this isoform may not lead to a decrease in 20-HETE production in renalblood vessels due to the presence of the CYP4F13 isoform in smallvessels and multiple CYP4F isoforms in large vessels.

In the present study, we observed numerous sex differences in thedistribution of CYP4A and CYP4F isoforms in different tubular andvascular segments. In the proximal tubule, male mice expressed theCYP4A10 and CYP4A12 isforms, while the CYP4A10 and CYP4A14 isoformswere detected in females. The expression of the CYP4A14 isoform, butnot the CYP4A12 isoform, in female mice under basal conditions is inagreement with previous studies using Northern blot analysis of wholekidney RNA ( 10 ). However, castration of male mice leads toa marked increase in the levels of the CYP4A14 and decrease in theCYP4A12 isoform in the kidney, suggesting a role for testosterone as anegative and positive regulator of these genes in mice. Both of thesechanges in expression can be prevented by treatment of mice withdihydrotestosterone (DHT) ( 10 ). Interestingly, renal20-HETE production is correlated with changes in the expression of theCYP4A12 isoform after castration and DHT treatment ( 10 ),suggesting that this isoform may be responsible for the higher rate of20-HETE production in the renal cortex of male vs. female mice.Previous studies have also detected increased levels of lauric acidhydroxylase activity and CYP4A protein in the kidneys of male vs.female mice ( 9 ).- z, ]# J* M! t8 y
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The only sex difference in distribution of the CYP4F isoforms was foundin the glomerulus. Glomeruli from male mice expressed the CYP4F13,CYP4F14, and CYP4F15 isoforms, whereas those from female mice expressedthe CYP4F13, CYP4F16, and CYP4F18 isoforms. Expression of the CYP4F15isoform in the glomeruli of male mice was the only segment in which theCYP4F15 isoform was detected in the present study. Interestingly, thisisoform could not be amplified from whole kidney RNA samples but couldbe readily amplified from the liver (Stec, unpublishedobservations). If the CYP4F15 isoform avidly metabolizes AA to20-HETE, the differential expression of CYP4F15 in the liver vs. thekidney may contribute to the present observation that the production of20-HETE is much greater in the liver vs. the kidney of C57BL/6J mice.This concept is consistent with the results of recent studiesindicating that the CYP4F2 is the primary isoform responsible for theproduction of 20-HETE in the human kidney ( 4, 16 ), whereasother human isoforms such as CYP4F8 and CYP4F12 appear to primarilymetabolize prostaglandins and leukotrienes ( 3, 19 ). Themurine CYP4F14, -15, -16, and -18 isoforms share a high degree of aminoacid homology with the CYP4F2 isoform (Table 3 ). Of these, the CYP4F16 isoform was the one detected in most vascular and tubular segments analyzed. However, at the present time the relative catalytic activity of the murine CYP4Fisoforms to metabolize AA vs. other substrates is unknown, so it isdifficult to speculate on which isoform is most important in producing20-HETE in various tubular and vascular segments of the murine kidney.
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Table 3. Amino acid homology among mouse, rat, and human CYP4A and 4F proteins& F* \5 ~) [; O$ P
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To determine the functional significance of the observed differences inCYP4A and CYP4F isoform distribution in male vs. female C57BL/6J mice,we measured the metabolism of AA by microsomes prepared from the cortexand outer medulla of the kidney. Despite the fact that we characterizedclear sex differences in the expression of CYP4A and CYP4F mRNA invarious nephron segments of the kidney, we did not detect anysignificant difference in the production of 20-HETE by microsomesprepared from the renal cortex or outer medulla of male and femaleC57BL/6J mice. The reason for this observation remains to bedetermined, but there are many possible explanations. For example,CYP4A and CYP4F proteins in the kidney may be different from theisoforms detected by RT-PCR in the present study. It is also possiblethat many of the murine isoforms do not metabolize AA to 20-HETE butare primarily involved in the hydroxylation of prostaglandins andleukotrienes in vivo.

Interestingly, we found that the production of 20-HETE was much lowerin the kidneys of mice compared with that reported in other species andabout five times lower than that seen in the livers of the same animals( 1, 14 ). The low level of 20-HETE production in thekidneys of C57BL/6J female mice is in agreement with previous reportsof wild-type CYP4A14 knockout mice on a 129 mixed genetic background( 10 ). Previous reports of renal 20-HETE production in malemice have indicated higher levels in both a mixed 129 geneticbackground ( 10 ) as well as in NMRI mice ( 11 ).Because we only examined distribution of the CYP4A and CYP4F isoformsin C57BL/6J mice in the present study, it is possible that theseobserved discrepancies in 20-HETE production may be due to differencesin the distribution of these isoforms between various inbred strains.The low level of metabolism of AA by renal microsomes from C57BL/6Jmice may indicate that AA is not the preferred substrate for the CYP4Aand CYP4F isoforms in vivo. This hypothesis is supported by previousstudies that have demonstrated much higher levels of lauric acidhydroxylation by renal microsomes compared with AA hydroxylation inother inbred strains of mice ( 9, 11, 27 ). Also, studieswith purified CYP4A14 protein indicate that this isoform is not able tometabolize AA but is able to efficiently metabolize lauric acid( 10 ). The CYP4A10 isoform shares 92% amino acid homologywith the rat CYP4A1 isoform, whereas the CYP4A12 and CYP4A14 isoformsshare the greatest homology with rat CYP4A8 and CYP4A3 isoforms,respectively (Table 2 ). The rat CYP4A1 isoform has been demonstrated tohave the highest 20-HETE-generating capability of the rat isoforms even though it is expressed at low levels in the kidney under basal conditions ( 18 ). However, despite the great degree ofhomology with the rat CYP4A isoforms, the specific ability of the mouse CYP4A10 and CYP4A12 isoforms to generate 20-HETE has yet to be reported.
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The mouse provides a powerful tool by which the various physiologicalactions of 20-HETE in the kidney can be cleanly dissected in vivo. Wehave identified the isoforms of the CYP4A and CYP4F families throughoutthe nephron and in renal vessels that may be responsible for theproduction of 20-HETE in vivo. Several differences in the distributionof CYP4A and CYP4F isoforms were identified in specific renal tubularand vascular segments analyzed in male and female C57BL/6J mice.Disparities in the CYP4A and CYP4F isoforms may account for the alteredlevels of renal 20-HETE production between various inbred strains ofmice as well as the difference in 20-HETE production between male andfemale mice. The specific substrate specificities of each of the murineCYP4A and CYP4F isoforms need to be determined to fully test thishypothesis. These strain differences in renal 20-HETE production needto be taken into account when the appropriate genetic background in which to alter the levels of renal 20-HETE production in mice is beingconsidered. Strains such as the C57BL/6J exhibit low levels of renal20-HETE production and would not be an ideal genetic background formodels to examine reduced renal 20-HETE production by gene knockout;however, this strain may be appropriate when the physiological effectsof increased renal 20-HETE production via cell-type-specific transgenictargeting are being examined. These types of transgenic andgene-targeted models will be critical to fully determine the complexrole of intrarenal 20-HETE production in the regulation of bloodpressure and renal function.% K8 q' v0 j3 X9 x! M

ACKNOWLEDGEMENTS, j9 P, N2 L1 G/ z& P/ {
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This work was supported by an American Heart Association, HeartlandAffiliate, Beginning-Grant-in-Aid (to D. E. Stec) and NationalHeart, Lung, and Blood Institute Grant HL-51971.
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