Molecular Cloning, Enzymatic Characterization, Developmental Expression, and Cellular Localization of a Mouse Cytochrome P450 Highly Expressed in Kidney*

Jixiang MaDagger , Wei QuDagger , Paula E. ScarboroughDagger , Kenneth B. Tomer§, Cindy R. Moomaw, Robert Maronpot, Linda S. Davisparallel , Matthew D. Breyerparallel , and Darryl C. ZeldinDagger **

From the Dagger  Laboratories of Pulmonary Pathobiology, § Structural Biology, and  Experimental Pathology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 and the parallel  Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232

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ABSTRACT
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EXPERIMENTAL PROCEDURES
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A cDNA encoding a new cytochrome P450 was isolated from a mouse liver library. Sequence analysis reveals that this 1,886-base pair cDNA encodes a 501-amino acid polypeptide that is 69-74% identical to CYP2J subfamily P450s and is designated CYP2J5. Recombinant CYP2J5 was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 cells using a baculovirus system. Microsomal fractions of CYP2J5/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolize arachidonic acid to 14,15-, 11,12-, and 8,9-epoxyeicosatrienoic acids and 11- and 15-hydroxyeicosatetraenoic acids (catalytic turnover, 4.5 nmol of product/nmol of cytochrome P450/min at 37 °C); thus CYP2J5 is enzymologically distinct. Northern analysis reveals that CYP2J5 transcripts are most abundant in mouse kidney and present at lower levels in liver. Immunoblotting using a polyclonal antibody against a CYP2J5-specific peptide detects a protein with the same electrophoretic mobility as recombinant CYP2J5 most abundantly in mouse kidney microsomes. CYP2J5 is regulated during development in a tissue-specific fashion. In the kidney, CYP2J5 is present before birth and reaches maximal levels at 2-4 weeks of age. In the liver, CYP2J5 is absent prenatally and during the early postnatal period, first appears at 1 week, and then remains relatively constant. Immunohistochemical staining of kidney sections with anti-human CYP2J2 IgG reveals that CYP2J protein(s) are present primarily in the proximal tubules and collecting ducts, sites where the epoxyeicosatrienoic acids are known to modulate fluid/electrolyte transport and mediate hormonal action. In situ hybridization confirms abundant CYP2J5 mRNA within tubules of the renal cortex and outer medulla. Epoxyeicosatrienoic acids are endogenous constituents of mouse kidney thus providing direct evidence for the in vivo metabolism of arachidonic acid by the mouse renal epoxygenase(s). Based on these data, we conclude that CYP2J5 is an enzymologically distinct, developmentally regulated, protein that is localized to specific nephron segments and contributes to the oxidation of endogenous renal arachidonic acid pools. In light of the well documented effects of epoxyeicosatrienoic acids in modulating renal tubular transport processes, we postulate that CYP2J5 products play important functional roles in the kidney.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Cytochrome P450s have been the subject of intense investigation by toxicologists and pharmacologists over the past 4 decades because they catalyze the oxidative, peroxidative, and reductive metabolism of a wide variety of drugs, industrial chemicals, environmental pollutants, and carcinogens (1, 2). Some of these enzymes also catalyze the NADPH-dependent oxidation of arachidonic acid to cis-epoxyeicosatrienoic acids (5,6-, 8,9-, 11,12-, and 14,15-EET),1 mid-chain cis-trans-conjugated dienols (5-, 8-, 9-, 11-, 12-, and 15-HETE), and/or omega -terminal alcohols of arachidonic acid (16-, 17-, 18, 19-, and 20-HETE) (3-5). A particular interest in the epoxygenase reaction has developed because the EETs are endogenous constituents of numerous tissues and because they possess a myriad of potent biological activities (3-7). For example, the EETs have been shown to control peptide hormone secretion in the pancreas, pituitary, and hypothalamus (8, 9), regulate vascular tone in the intestine and brain (10, 11), affect ion transport and airway smooth muscle tone in the lung (12, 13), and improve functional recovery following global ischemia in the heart (14).

P450-derived eicosanoids have been extensively studied in the kidney and have been shown to substantially contribute to integrated renal function (6, 7). Thus, the EETs and/or their hydration products (the DHETs) inhibit Na+ transport and mediate angiotensin II-induced rises in cytosolic Ca2+ in the proximal tubule (15, 16), stimulate prostaglandin synthesis, and inhibit Na+ reabsorption, K+ secretion, and vasopressin-stimulated water reabsorption in the collecting duct (17-19), activate Na+/H+ exchange, amplify vasopressin-induced increases in cytosolic Ca2+, and stimulate cellular proliferation in renal glomerular mesangial cells (20, 21), modulate renal Na+/K+-ATPase (22), affect intrarenal vascular tone, and renal vascular smooth muscle cell K+ channel activity (23-25), and inhibit renal cortical renin release (26). Recent studies demonstrating that (a) the rat renal epoxygenase(s) are under regulatory control by dietary salt, (b) clotrimazole inhibition of the rat renal epoxygenase(s) leads to the development of salt-dependent hypertension, (c) the salt-sensitive phenotype in the Dahl rat model of genetic hypertension is associated with decreased renal microsomal arachidonic acid epoxygenase activity and an inability to up-regulate this activity in response to salt loading, (d) the developmental phase of hypertension in spontaneously hypertensive rats is associated with alterations in kidney P450 arachidonic acid metabolism, and (e) the urinary excretion of epoxygenase metabolites is increased during pregnancy-induced hypertension in humans have supported the hypothesis that P450-derived arachidonic acid metabolites may be involved in the pathophysiology of hypertension (6, 7, 27-32). Together, these findings suggest that the renal P450 arachidonic acid monooxygenases may be important in controlling blood pressure and body fluid volume/composition and may be part of an adaptive response of the kidney to excess dietary salt intake (6, 7).

Despite considerable investigations into the physiological aspects of the renal epoxygenase pathway, little is known with regard to the biochemical and molecular properties of the renal P450 enzyme(s) active in the biosynthesis of the EETs. A member of the CYP2C subfamily was purified from rabbit kidney and shown to catalyze arachidonic acid epoxidation and omega /omega -1 hydroxylation (33). Immunochemical studies suggested that CYP2C isoforms were responsible for some of the arachidonic acid epoxygenase activity present in rat and rabbit kidney microsomal fractions (27, 33). Work by Karara et al. (34) and Imaoka et al. (35) demonstrated that CYP2C23 was highly expressed in rat kidney and active in the regio- and stereoselective epoxidation of arachidonic acid (34, 35). CYP2C8, cloned from a human kidney cDNA library, was also an active arachidonic acid epoxygenase; however, this P450 was expressed at relatively low levels in human kidney (36, 37). More recently, our laboratory identified a human P450 of the CYP2J subfamily that was highly expressed in a number of extrahepatic tissues including the kidney and active in the metabolism of arachidonic acid to EETs (38). However, studies on the regulation, importance, and functional role of this new human P450 epoxygenase in the kidney have not been possible because of the limited availability of fresh, histologically normal and abnormal human kidney tissues. Herein, we report the cDNA cloning and heterologous expression of CYP2J5, a new mouse P450 that is abundant in the kidney and active in the epoxidation and midchain hydroxylation of arachidonic acid. We show that this heme-thiolate protein is regulated during development and in a tissue-specific fashion. We further demonstrate, using immunohistochemical techniques and in situ hybridization, that CYP2J5 mRNA and protein are localized to sites within the nephron where the EETs are known to affect fluid/electrolyte transport and mediate hormonal action.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Materials-- [alpha -32P]dATP and [1-14C]arachidonic acid were purchased from NEN Life Science Products. Restriction enzymes were purchased from New England Biolabs (Beverly, MA). PCR reagents including AmpliTaq® DNA polymerase were purchased from Perkin-Elmer. Triphenylphosphine, alpha -bromo-2,3,4,5,6-pentafluorotoluene, N,N-diisopropylethylamine, N,N-dimethylformamide, and diazald were purchased from Aldrich. All other chemicals and reagents were purchased from Sigma unless otherwise specified.

Screening of the cDNA Library-- An oligo(dT)-primed Lambda ZAP B6/CBAF1J male mouse liver cDNA library (Stratagene, La Jolla, CA) was screened with the 1.8-kb CYP2J3 cDNA probe under conditions identical to those described previously (14). Approximately 100 duplicate positive clones were identified, of which 21, selected at random, were plaque-purified and rescued into pBluescript SK(+) (Stratagene). Plasmid DNAs were isolated using a Qiagen Plasmid Purification Kit (Qiagen Inc., Chatsworth, CA), and insert sizes were determined by agarose gel electrophoresis after EcoRI digestion. The pBluescript cDNA inserts were partially sequenced by the dideoxy chain termination method using Sequenase version 2.0 (U. S. Biochemical Corp.) and T3/T7 oligonucleotide primers. Nucleotide sequences were analyzed by searching GenBankTM and EMBL data bases utilizing GCG software (Genetics Computer Group, Inc., Madison, WI). Sixteen of the duplicate positive clones contained sequences that were identical and shared homology with several human and rodent CYP2 family P450s (1). One of these clones (clone JM-6)2 was completely sequenced utilizing a total of 16 oligonucleotide primers (21-25 nucleotides, each) that spanned the entire length of the sense and antisense cDNA strands. Oligonucleotides were synthesized and purified as described (14).

Heterologous Expression of Recombinant CYP2J5-- Co-expression of the protein encoded by the cloned 1.886-kb JM-6 cDNA insert (CYP2J5) with CYPOR in Sf9 insect cells was accomplished with the pAcUW51-CYPOR shuttle vector (kindly provided by Dr. Cosette Serabjit-Singh, Glaxo Wellcome, Research Triangle Park, NC) and the BaculoGold Baculovirus Expression System (PharMingen, San Diego, CA) using methods similar to those previously described (14, 39). The CYP2J5 cDNA (nucleotides 42-1886) including the entire coding region was ligated into a slightly modified pAcUW51-CYPOR vector, and the orientation and identity of the resulting expression vector (pAcUW51-CYPOR-CYP2J5) were confirmed by sequence analysis. In this construct, the expression of CYPOR was controlled by the p10 promoter, whereas the expression of CYP2J5 was independently controlled by the polyhedrin promoter. Cultured Sf9 cells were co-transfected with the pAcUw51-CYPOR-CYP2J5 vector and linear wild-type BaculoGold viral DNA, and recombinant viruses were purified as described (14). Cultured Sf9 cells grown in spinner flasks at a density of 1.5-2 × 106 cells/ml were then infected with high titer CYP2J5-CYPOR recombinant baculovirus stock in the presence of delta -aminolevulinic acid and iron citrate (100 µM each). Cells co-expressing recombinant CYP2J5 and CYPOR were harvested 72 h after infection, washed twice with phosphate-buffered saline, and were used to prepare microsomal fractions by differential centrifugation at 4 °C as described previously (12). P450 content was determined spectrally according to the method of Omura and Sato (40) using a Shimadzu UV-3000 dual-wavelength/double-beam spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD). CYP2J5 protein was also expressed without CYPOR in Sf9 cells using the pBlueBacIV expression vector (Invitrogen, San Diego, CA). Sf9 cells expressing recombinant CYPOR but not P450 were prepared as described (39).

Incubations of Recombinant CYP2J5 with Arachidonic Acid, Product Characterization-- Reaction mixtures containing 0.05 M Tris-Cl buffer (pH 7.5), 0.15 M KCl, 0.01 M MgCl2, 8 mM sodium isocitrate, 0.5 IU/ml isocitrate dehydrogenase, 0.1-0.2 mg of CYP2J5-CYPOR-transfected Sf9 cell microsomal protein/ml, and [1-14C]arachidonic acid (25-55 µCi/µmol; 100 µM, final concentration) were constantly stirred at 37 °C. After temperature equilibration, NADPH (1 mM, final concentration) was added to initiate the reaction. At different time points, aliquots were withdrawn, and the reaction products were extracted and analyzed by reverse-phase HPLC as described (41). Products were identified by comparing their reverse- and normal-phase HPLC properties with those of authentic standards and by GC/MS (42, 43). For kinetic experiments, arachidonic acid concentrations were varied from 5 to 100 µM, and reactions were only allowed to proceed for 2-3 min to ensure that the quantitative assessment of the rates of product formation reflected initial rates. For chiral analysis, the EETs were collected from the HPLC eluent, derivatized, and resolved into the corresponding antipodes by chiral-phase HPLC as described previously (44, 45). Control studies were performed by incubating uninfected Sf9 cell microsomes and baculovirus-infected Sf9 cell microsomes expressing recombinant CYPOR but containing no spectrally evident P450 with arachidonic acid under identical conditions.

Detection of Endogenous EETs in Mouse Kidney-- Methods used to quantify endogenous EETs present in mouse kidney were similar to those used to quantify EETs in rat liver and heart (14, 46). Briefly, kidneys were frozen in liquid nitrogen and homogenized in phosphate-buffered saline containing triphenylphosphine. The homogenate was extracted as described (14), and the combined organic phases were evaporated in tubes containing mixtures of [1-14C]8,9-EET, [1-14C]11,12-EET, and [1-14C]14,15-EET internal standards (56-57 µCi/µmol, 80 ng each). Saponification, silica column purification, HPLC resolution, GC/MS analysis, and quantification were done as described elsewhere (14, 45, 46).

Northern Blot Hybridization and RNA PCR Analysis-- Normal mouse tissues were obtained from adult male C57BL/6J mice fed NIH 31 rodent chow (Agway, St. Mary, OH) ad libitum and sacrificed by lethal CO2 inhalation. RNA was prepared by the guanidinium thiocyanate/cesium chloride density gradient centrifugation method as described previously (47). Total RNA (25 µg) prepared from mouse testes, small intestine, muscle, lung, liver, kidney, heart, colon, and brain was denatured and electrophoresed in 1.2% agarose gels containing 2.2 M formaldehyde. After capillary pressure transfer to Hybond-N+ membranes (Amersham Pharmacia Biotech), the blots were hybridized with either the cloned 1.886-kb JM-6 cDNA insert or with the following JM-6 sequence-specific oligonucleotide probe (5'-TCAAAAACATAATCATATTTGGGATCCTTGCACAC-3', complementary to nucleotides 23-57 of the JM-6 cDNA). With the cDNA probe, hybridizations were performed at 42 °C in 50% formamide containing 0.7 M NaCl, 70 mM sodium citrate, 5× Denhardt's solution, 0.5% (w/v) SDS, and 0.1 mg of heatdenatured salmon sperm DNA/ml. With the oligonucleotide probe, hybridizations were performed at 50 °C in 10% formamide containing 0.9 M NaCl, 90 mM sodium citrate, 2× Denhardt's solution, 0.5% (w/v) SDS, and 0.1 mg of heat-denatured salmon sperm DNA/ml. The double-stranded cDNA probe was labeled with [alpha -32P]dATP by nick translation using Escherichia coli polymerase I. The oligonucleotide probe was end-labeled using T4 polynucleotide kinase and [gamma -32P]ATP. Northern blot results were confirmed by PCR amplification of reversetranscribed mouse RNAs using the GeneAmp® RNA PCR Kit (Perkin-Elmer). The following JM-6 sequence-specific oligonucleotides were used: forward primer, 5'-ATCAGAGAAGCGAAAAGAATGTAG-3', corresponding to nucleotides 1549-1572 of the JM-6 cDNA; reverse primer, 5'-CCATTTCCTCTGATTCTGACTCAT-3', complementary to nucleotides 1810-1833 of the JM-6 cDNA. Reverse transcription was performed with 1 µg of total RNA in a buffer containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 2.5 µM random hexamers, 1 mM each of dGTP, dATP, dTTP, and dCTP, and 50 units of Moloney murine leukemia virus reverse transcriptase incubated at 42 °C for 1 h. The PCR amplifications were performed in the presence of 2 mM MgCl2, 0.15 µM forward and reverse primers, and 2.5 units of AmpliTaq® DNA polymerase. Following an initial incubation for 105 s at 95 °C, samples were subjected to 35 cycles of 15 s at 95 °C and 30 s at 62 °C. The PCR products were electrophoresed on 1.2% agarose gels containing ethidium bromide. The following beta -actin sequence-specific primers were used to control for the quality and amount of RNA: forward primer, 5'-GAGCTATGAGCTGCCTGACG-3', corresponding to nucleotides 776-795 of the mouse beta -actin cDNA; reverse primer, 5'-AGCACTTGCGGTGCACGATG-3', complementary to nucleotides 1166-1185 of the mouse beta -actin cDNA.

Protein Immunoblotting and Immunohistochemistry-- Microsomal fractions were prepared from adult male C57BL/6J mouse tissues by differential centrifugation at 4 °C as described previously (12). Polyclonal antibodies against the purified, recombinant human CYP2J2 proteins (anti-CYP2J2 IgG) were raised in New Zealand White rabbits and affinity purified as described previously (38). A peptide corresponding to amino acids 103-117 of the deduced CYP2J5 sequence was designed based upon sequence alignments with known CYP2 family P450s. The peptide was synthesized and HPLC-purified by the Protein Chemistry Facility at the University of North Carolina (Chapel Hill, NC). The peptide was coupled to keyhole limpet hemocyanin via a carboxyl-terminal cysteine to enhance antigenicity. Polyclonal antibodies against this CYP2J5-specific peptide (anti-CYP2J5pep IgG) were raised in New Zealand White rabbits using previously described methods (48). Purified recombinant human CYP2J2 was prepared as described previously (38). Microsomes prepared from Sf9 insect cells infected with recombinant CYP2J6 baculovirus3 were used as a source of mouse CYP2J6. Purified preparations of mouse CYP2A4 and CYP2A5 were a gift from Dr. Masahiko Negishi (NIEHS, National Institutes of Health). Purified recombinant CYP2C29, CYP2C37, CYP2C38, CYP2C39, and CYP2C40 were a gift from Dr. Joyce Goldstein (NIEHS). For immunoblotting, microsomal fractions or purified P450s were electrophoresed in SDS-10% (w/v) polyacrylamide gels (80 × 80 × 1 mm), and the resolved proteins were transferred electrophoretically onto nitrocellulose membranes. Membranes were immunoblotted using either rabbit anti-CYP2J2 IgG or rabbit anti-CYP2J5pep IgG, goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad), and the ECL Western blotting Detection System (Amersham Pharmacia Biotech) as described (38). Protein determinations were performed according to the method of Bradford (49). Preimmune serum, collected from the rabbits prior to immunization, did not cross-react with CYP2J5 or with microsomal fractions prepared from mouse tissues.

For immunohistochemistry, adult mouse, rat, and human kidney tissues were fixed in 10% neutral buffered formalin, processed routinely, and embedded in paraffin. Normal human kidney was obtained through the Cooperative Human Tissue Network (National Disease Research Interchange, Philadelphia, PA). Normal rat and mouse kidneys were obtained from adult male Fischer 344 rats and C57BL/6J mice that had been fed ad libitum and killed by lethal CO2 inhalation. Localization of CYP2J protein expression was investigated on serial kidney sections (5-6 µm) with the anti-CYP2J2 IgG (1:100 dilution) using methods previously described for heart and intestine (14, 41). Preimmune rabbit IgG was used as the negative control in place of the primary antibody.

In Situ Hybridization-- Radiolabeled antisense and sense RNA probes to mouse CYP2J5 were transcribed from the linearized CYP2J5 (JM-6)/pBluescript construct using the T7 and T3 RNA polymerases and [alpha -35S]UTP. The 35S-labeled probes were then subjected to alkaline hydrolysis. In situ hybridization was performed on 4% paraformaldehyde-fixed, paraffin-embedded mouse kidney sections as described previously (50). Briefly, 6-7-µm sections were deparaffinized, refixed in paraformaldehyde, treated with proteinase K (20 µg/ml), washed with phosphate-buffered saline, refixed in paraformaldehyde, treated with triethanolamine plus acetic anhydride (0.25% v/v), and then dehydrated with 100% ethanol. Sense or antisense RNA probes were hybridized to the sections at 55 °C for approximately 18 h. After hybridization, the sections were washed once in 5× SSC plus 10 mM beta -mercaptoethanol for 30 min and once more in 50% formamide, 2× SSC, and 100 mM beta -mercaptoethanol for 60 min at 65 °C. After two additional washes in 10 mM Tris-Cl, 5 mM EDTA, 500 mM NaCl (TEN) at 37 °C, sections were treated with RNase A (10 µg/ml) at 37 °C for 30 min, followed by another wash in TEN at 37 °C. Sections were then washed twice in 2× SSC and twice more in 0.1× SSC at 65 °C. Slides were dehydrated with graded ethanol containing 300 mM ammonium acetate, dipped in photoemulsion (Ilford K5, Knutsford, Cheshire, UK) diluted 1:1 with 2% glycerol, and exposed for 4-5 days at 4 °C. After developing in Kodak D-19, slides were counterstained with hematoxylin.

Developmental Regulation of CYP2J5-- Kidneys and livers from male and female C57BL/6J mice ages 18 days fetal to 16 weeks postnatal (adult) were obtained from Hilltop Labs (Scottdale, PA). Animals were weaned at 3 weeks of age and then fed NIH 31 rodent chow (Agway, St. Mary, OH) ad libitum and sacrificed by lethal CO2 inhalation. Tissues were frozen in liquid nitrogen immediately following collection and shipped on dry ice. Microsomes were prepared from pooled frozen tissues obtained from 3 to 6 animals at each age, and protein immunoblotting was performed as detailed above.

Synthetic Procedures-- The [1-14C]EET internal standards were synthesized from [1-14C]arachidonic acid (56-57 µCi/µmol) by nonselective epoxidation as described previously (51). EETs and HETE standards were prepared by total chemical synthesis according to published procedures (52-54). DHETs and [1-14C]DHETs were prepared by chemical hydration of individual EETs as described (55). All synthetic compounds were purified by reverse-phase HPLC (42). Methylations were performed using an ethereal solution of diazomethane (56). Pentafluorobenzyl esters were formed by reaction with pentafluorobenzyl bromide as described (46).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Cloning of Mouse CYP2J5 cDNA-- Screening of the mouse liver cDNA library with the rat CYP2J3 cDNA probe yielded 16 duplicate positive clones that contained identical sequences. One of these clones (clone JM-6) was selected for further study. Complete nucleic acid sequence analysis of clone JM-6 revealed that the cDNA was 1886 nucleotides long, contained an open reading frame between nucleotides 42 and 1544 flanked by initiation (ATG) and termination (TGA) codons, had a short 5'-untranslated region, and had a 342-nucleotide 3'-untranslated region with a polyadenylation tail (Fig. 1). The cDNA encoded a 501-amino acid polypeptide that had a derived molecular mass of 57,784 Da. The deduced amino acid sequence for JM-6 contained a putative heme binding peptide (FSMGKRACLGEQLA) with the underlined conserved residues and the invariant cysteine at position 447. The polypeptide encoded by JM-6 also contained other structural features associated with CYP2 family P450s including an amino-terminal hydrophobic peptide and a proline cluster between residues 40 and 51 (Fig. 1). A comparison of the deduced JM-6 amino acid sequence with that of other P450s indicated that it was (a) <32% identical to members of the CYP1, CYP3, CYP4, CYP5, CYP6, and CYP7 families, (b) 38-44% identical with several CYP2 family P450s, and (c) 69-74% identical to human, rabbit, and rat CYP2J P450s (1, 14, 38, 57, 58). Based on the amino acid sequence homology with other CYP2Js, the new mouse hemoprotein was designated CYP2J5 by the Committee on Standardized Cytochrome P450 Nomenclature (1).


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Fig. 1.   Nucleotide sequences for CYP2J5. The putative heme-binding peptide is underlined, and the conserved residues are in bold. An asterisk marks the termination codon. The location of the peptide used to prepare the anti-CYP2J5pep IgG is enclosed within a box.

Heterologous Expression and Enzymatic Characterization of Recombinant CYP2J5-- The protein encoded by clone JM-6 (CYP2J5) was expressed with/without CYPOR in Sf9 insect cells using the baculovirus expression system according to previously described methods (14, 38, 39). The level of expression of recombinant CYP2J5 was 10-15 nmol of P450/liter of infected Sf9 cells using the pAcUW51-CYPOR-CYP2J5 vector. CYP2J5 expression was significantly higher (~100 nmol of P450/liter of infected Sf9 cells) in the absence of CYPOR coexpression using the pBlueBacIV vector. Coomassie Brilliant Blue-stained SDS-polyacrylamide gels of lysates prepared from CYP2J5 baculovirus-infected Sf9 cells revealed a prominent band with an estimated molecular mass of 57-58 kDa that was not present in uninfected insect cell lysates (data not shown).

Microsomal fractions prepared from Sf9 insect cells co-expressing CYP2J5 and CYPOR metabolized arachidonic acid to EETs and midchain HETEs as the principal reaction products (catalytic turnover, 4.5 nmol of products/nmol of P450/min at 100 µM arachidonic acid) (Fig. 2A). We identified these metabolites by comparing their reverse- and normal-phase HPLC properties with those of authentic standards and by GC/MS analysis. None of the metabolites were formed when NADPH was omitted from the reaction. Furthermore, uninfected Sf9 insect cell microsomes (data not shown) and baculovirus-infected Sf9 cell microsomes expressing recombinant CYPOR but containing no spectrally evident P450 (Fig. 2B) did not metabolize arachidonic acid to EETs or midchain HETEs. This indicated that nonspecific oxidation from sources other than P450 was minimal and demonstrated that the eicosanoid products were generated in a P450-dependent fashion. Thus, based on the chromatogram in Fig. 2, we conclude that CYP2J5 is both an arachidonic acid epoxygenase and an arachidonic acid midchain hydroxylase.



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Fig. 2.   Metabolism of arachidonic acid by recombinant CYP2J5. A, reverse-phase HPLC chromatogram of the organic soluble metabolites generated during an incubation of Sf9 insect cell microsomes containing recombinant CYP2J5 and CYPOR with [1-14C]arachidonic acid. Chromatogram is representative of 15 separate experiments. B, reverse-phase HPLC chromatogram of the organic soluble metabolites generated during incubation of Sf9 insect cell microsomes containing CYPOR but not containing P450 with [1-14C]arachidonic acid. Results are representative of 5 separate experiments. C, plot of reaction velocity versus substrate concentration. The arachidonic acid concentration was varied from 5 to 100 µM, and reactions were allowed to proceed for 2-3 min to ensure that the quantitative assessment of the rates of product formation reflected initial rates. D, Lineweaver-Burk transformation of the data shown in C. The apparent Km and Vmax values were derived by fitting the results to the Michaelis-Menten equation. Results are representative of 7 separate experiments.

Regiochemical analysis of the EETs, which accounted for approximately half of the reaction products, revealed a preference for epoxidation at the 14,15-olefin (64% of total EETs) as evidenced by the recovery of 14,15-EET and its hydration product, 14,15-DHET (Fig. 2A, Table I). Epoxidation at the 11,12- and 8,9-olefins occurred less often (24 and 11% of total EETs, respectively), whereas epoxidation at the 5,6-olefin occurred only rarely (1% of the total EETs) (Table I). Stereochemical analysis of CYP2J5-derived EETs revealed a preference for (14S,15R)-, (11R,12S)-, and (8R,9S)-EETs (optical purity, 60, 69, and 58%, respectively) (Table I). Regiochemical analysis of the midchain hydroxylase products revealed a preference for 15- and 11-HETEs (26 and 15% of total HETEs, respectively). Hydroxylation at the 12-, 9-, 8-, and 5-positions occurred less often. Thus, CYP2J5 is enzymologically distinct from other CYP2J P450s in that (a) arachidonic acid epoxidation occurs with greater regioselectivity, and (b) it is the only CYP2J isoform that produces appreciable quantities of midchain HETEs.

                              
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Table I
Regio- and stereochemical composition of EETs produced by recombinant CYP2J5
The activity of recombinant CYP2J5 was reconstituted in the presence of NADPH and an NADPH-regenerating system as described under "Experimental Procedures." After 30 min, the EET products were extracted into ethyl ether, resolved into individual regioisomers by reverse- and normal-phase HPLC, derivatized to corresponding EET-PFB or EET-methyl esters, purified by normal-phase HPLC, and resolved into the corresponding antipodes by chiral-phase HPLC. Values shown are averages of at least four different experiments with S.E. <5%.

CYP2J5 containing microsomes metabolized arachidonic acid in a time-dependent manner with linear reaction kinetics up to 10-min incubation times at P450 concentrations up to 500 pmol/ml. 14,15-EET, the major product, was formed at a rate of 1.3 nmol of EET/nmol of P450/min. 11,12- and 8,9-EETs were formed at lower rates (0.5 and 0.2 nmol of EET/nmol of P450/min, respectively), whereas epoxidation at the 5,6-olefin occurred very slowly (<0.02 nmol EET/nmol P450/min). 15- and 11-HETEs, the major hydroxylase products, were formed at rates of 0.7 and 0.4 nmol of HETE/nmol of P450/min, respectively. Other midchain HETEs were formed at lower rates. A more detailed kinetic analysis revealed that the metabolism of arachidonic acid seemed to exhibit simple Michaelis-Menten kinetics over the range of substrate concentrations used (5-100 µM) (Fig. 2C). The Lineweaver-Burke double-reciprocal plot shown in Fig. 2D was used to derive an apparent Km of 83 µM and Vmax of 10.9 nmol of product/nmol of P450/min for CYP2J5.

Tissue Distribution of CYP2J5 mRNA and Protein-- To ascertain the relative organ abundance of CYP2J5 transcripts, total RNA extracted from various mouse tissues was blot hybridized under high stringency conditions with the full-length CYP2J5 cDNA probe. As shown in Fig. 3A, the CYP2J5 cDNA hybridized with kidney RNA to produce three transcripts as follows: (a) an abundant 1.8-2.0-kb transcript consistent with the size of the CYP2J5 mRNA; (b) a larger 3.2-kb transcript of similar intensity; and (c) a 4.2-kb transcript of significantly lower intensity. The identities of the 3.2- and 4.2-kb transcripts remain unknown, but these larger transcripts may represent other mouse P450s that share nucleic acid sequence homology with CYP2J5 or alternate splice variants of CYP2J5. The 1.9- and 3.2-kb CYP2J5 transcripts were also present at lower levels in mouse liver but were absent from other mouse tissues including testes, small intestine, skeletal muscle, lung, heart, colon, and brain (Fig. 3A). The differences in mRNA abundance were not due to differences in the amount of RNA applied to each lane as assessed by ethidium bromide staining of the gel prior to transfer (Fig. 3A) and the membrane following transfer (data not shown). Identical results were obtained using a CYP2J5 sequence-specific oligonucleotide probe (data not shown). To confirm independently the narrow tissue distribution of CYP2J5 mRNAs, we used a sensitive and specific RNA PCR method to amplify a 284-base pair DNA fragment (predicted size) from reverse-transcribed mouse kidney and liver RNA (Fig. 3B). We were unable to amplify a fragment from testis, small intestine, skeletal muscle, lung, heart, colon, or brain RNA indicating that CYP2J5 mRNA expression was below the limit of detection of the RNA PCR assay in these tissues (Fig. 3B). RNA-PCR analysis using sequence-specific primers for beta -actin confirmed that the observations were not due to differences in the quality or amount of RNA used (Fig. 3B). Thus, based upon the Northern analysis and RNA PCR, we conclude that CYP2J5 mRNA is primarily present in mouse kidney, expressed at lower levels in mouse liver, and absent from other mouse tissues.


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Fig. 3.   Tissue distribution of CYP2J5 mRNAs by Northern analysis and RNA PCR. A, total RNA (25 µg) isolated from various mouse tissues was denatured and electrophoresed in a 1.2% agarose gel containing 2.2 M formaldehyde. After capillary-pressure transfer to Hybond-N+ membranes, the blot was hybridized with the cloned 1.886-kb CYP2J5 cDNA labeled with [alpha -32P]dATP by nick translation. Top, autoradiograph of blot after 24-h exposure time. Bottom, ethidium bromide-stained gel before transfer. B, total RNA (1 µg) was reverse-transcribed using random hexamers and Moloney murine leukemia virus reverse transcriptase as described under "Experimental Procedures." PCR amplifications were then performed using either CYP2J5 or mouse beta -actin sequence-specific oligonucleotide primers and AmpliTaq® DNA polymerase as described. The PCR products were electrophoresed on 1.2% agarose gels containing ethidium bromide. Top, ethidium bromide-stained gel of DNAs amplified using CYP2J5-specific primers. Bottom, ethidium bromide-stained gel of DNAs amplified using beta -actin-specific primers.

Immunoblotting of microsomal fractions prepared from Sf9 insect cells infected with recombinant CYP2J5 baculovirus stock using either the anti-CYP2J2 IgG (Fig. 4A) or the anti-CYP2J5pep IgG (Fig. 4B) showed a primary band at approximately 57-58 kDa indicating that both of these antibodies cross-reacted with mouse CYP2J5. Control studies demonstrated that the anti-CYP2J5pep IgG did not cross-react with uninfected Sf9 insect cell microsomes or with the following recombinant mouse P450s: CYP2J6, CYP2A4, CYP2A5, CYP2C29, CYP2C37, CYP2C38, CYP2C39, and CYP2C40 (Fig. 4B). The anti-CYP2J2 IgG, which has been previously shown to immunoreact with CYP2J2 and CYP2J3 (14, 38), also immunoreacted with recombinant CYP2J6 but did not react with uninfected Sf9 insect cell microsomes (Fig. 4A). In order to examine the tissue distribution of CYP2J5 protein, we performed immunoblotting of microsomal fractions prepared from various mouse tissues. As shown in Fig. 4C, anti-CYP2J5pep IgG detected a protein with the same electrophoretic mobility as recombinant CYP2J5 in mouse kidney microsomes and, at lower levels, in mouse liver microsomes. This band was not present in microsomal fractions prepared from other mouse tissues including stomach, small intestine, skeletal muscle, lung, heart, colon, and brain (Fig. 4C). There was little inter-animal variation in the tissue expression of CYP2J5 protein (data not shown). The CYP2J5 peptide antibody also immunoreacted, albeit less intensely, with two higher molecular mass bands (~66-67 and ~75-76 kDa) (Fig. 4C). Although the identity of these additional immunoreactive bands that were present in virtually all tissues tested remains unknown, it is unlikely that they represent P450s based upon their molecular mass. To confirm the tissue distribution of CYP2J5 protein, we immunoblotted mouse microsomes with the anti-CYP2J2 IgG. As in the case of the anti-CYP2J5pep IgG, we detected a protein with the same electrophoretic mobility as recombinant CYP2J5 predominantly in mouse kidney and, at lower levels, in mouse liver microsomes (data not shown). Based on these data, we concluded that CYP2J5 mRNA and protein are abundant in mouse kidney, present at lower levels in liver, and absent from other mouse tissues. Importantly, CYP2J5 is the only mouse P450 arachidonic acid epoxygenase known to be expressed predominantly in the kidney.


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Fig. 4.   Tissue-specific expression of CYP2J5 by protein immunoblotting. A, microsomal fractions prepared from uninfected Sf9 insect cells or insect cells infected with recombinant CYP2J5 or CYP2J6 baculovirus stock (0.5 pmol of P450/lane) were electrophoresed on SDS-10% polyacrylamide gels and the resolved proteins transferred to nitrocellulose membranes. Membranes were immunoblotted with the anti-human CYP2J2 IgG and goat anti-rabbit IgG conjugated to horseradish peroxidase as described under "Experimental Procedures." The immunoreactive proteins were then visualized using the ECL detection system and autoradiography. B, purified recombinant CYP2J2, CYP2A4, CYP2A5, CYP2C29, CYP2C37, CYP2C38, CYP2C29, or CYP2C40, microsomal fractions prepared from Sf9 cells infected with recombinant CYP2J5 or CYP2J6 baculovirus stock (0.5 pmol P450/lane), or microsomes prepared from uninfected Sf9 cells were electrophoresed, transferred to nitrocellulose, and immunoblotted with the anti-CYP2J5pep IgG as described. C, microsomal fractions prepared from mouse stomach, small intestine, muscle, lung, liver, kidney, heart, colon, brain (25 µg of microsomal protein/lane), or Sf9 cells infected with recombinant CYP2J5 or CYP2J6 baculovirus stock (0.25 pmol P450/lane) were electrophoresed, transferred to nitrocellulose, and immunoblotted with the anti-CYP2J5pep IgG as described.

Immunolocalization of CYP2J Protein Expression in the Kidney-- To determine the distribution of CYP2J protein(s) within the kidney, we stained formalin-fixed paraffin-embedded kidney sections with the anti-CYP2J2 IgG. Similar staining patterns were observed in mouse, rat, and human kidney. As shown in Fig. 5, CYP2J protein expression was most abundant in the renal cortex, the outer stripe of the outer medulla, and the renal papilla. CYP2J immunopositivity was generally intense throughout the proximal tubules; however, staining in the straight part of the proximal tubules (corresponding to the P2/P3 segments) was generally more intense than in the convoluted part (corresponding to the P1 segment) (Fig. 5, B, C, D, and H). Within the collecting ducts, immunostaining was most intense in the papillary collecting ducts and was present at lower levels in the inner and outer medullary collecting ducts (Fig. 5, B, E, and F). Strong positive immunostaining was present in the transitional epithelium lining the renal pelvis (Fig. 5F). Immunoreactivity was also present, albeit in significantly lower amounts, in distal tubules (including the thick ascending limb and distal convoluted tubule), and was generally absent from vascular structures, glomeruli, the thin limbs of Henle's loop, and intervening stromal tissue (Fig. 5, C, D, F, G, and H). Preimmune IgG produced negative staining throughout the kidney (Fig. 5A). The immunolocalization of CYP2J protein(s) to regions of the nephron where energy-dependent absorption and secretion take place has potentially important functional implications given the well known effects of EETs and HETEs in modulating fluid and electrolyte transport in the renal proximal tubules and collecting ducts (6, 7, 15-19, 22). To our knowledge, this report is the first to localize expression of a P450 epoxygenase to these portions of the nephron.


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Fig. 5.   Immunohistochemical localization of CYP2J in mouse, rat, and human kidney. A, mouse kidney section stained with preimmune IgG showing absence of positive immunostaining (negative control). B, adjacent mouse kidney section stained with anti-human CYP2J2 IgG showing positive immunostaining (brown color) in proximal convoluted tubules in the cortex (C) and the straight segments of the proximal tubules in the outer stripe (O) of the outer medulla. Tubules in the inner stripe (I) of the outer medulla are largely negative for CYP2J immunoreactivity. C, higher magnification of central portion of B. D, higher magnification of upper left portion of C showing strong positive staining in proximal convoluted tubules, minimal staining in distal tubules (asterisks), and negative staining in an arcuate artery (A). E, rat kidney section stained with anti-human CYP2J2 IgG showing positive brown immunostaining of the collecting ducts in the renal papilla. F, renal papilla (P) from another rat showing positive immunostaining of collecting ducts and pelvic transitional epithelium (arrowheads). G, higher magnification of the large renal artery shown in E demonstrating an absence of CYP2J immunostaining in both vascular smooth muscle and endothelium. H, human kidney section showing positive immunostaining in proximal convoluted tubules in renal cortex with minimal staining of distal tubules (asterisks) and negative staining in an arcuate artery (A). Magnifications × 33 (A, B, and E), 82 (C and F), and 164 (D, G, and H).

Localization of CYP2J5 mRNA by in Situ Hybridization-- To ascertain the distribution of CYP2J5 mRNAs within the kidney, we performed in situ hybridization of paraformaldehyde-fixed, paraffin-embedded mouse kidney sections with the 35S-labeled CYP2J5 antisense RNA probe. As shown in Fig. 6, CYP2J5 mRNAs were primarily localized to the renal cortex and the outer stripe of the outer medulla. Intense labeling was observed in both the convoluted and the straight portions of the proximal tubules (Fig. 6, B-D). No significant labeling was present in glomeruli, renal blood vessels, the thin limb of Henle's loop, or the thick ascending limb. Interestingly, distal portions of the nephron (including the distal convoluted tubules and collecting ducts) did not significantly label with the CYP2J5 RNA probe (Fig. 6, B and C), despite positive staining of these regions with the anti-CYP2J2 IgG which cross-reacts with CYP2J5 and other CYP2J proteins (Figs. 4 and 5). Hybridization of kidney sections with the sense RNA probe yielded no appreciable staining (Fig. 6A). Therefore, in situ hybridization confirms that, like CYP2J protein(s), CYP2J5 mRNAs are abundant within the proximal tubules; however, the CYP2J immunostaining observed within distal nephron segments may be due to the presence of other CYP2J proteins in this region or, alternatively, may reflect inherent differences in CYP2J5 mRNA and protein stability.


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Fig. 6.   Distribution of CYP2J5 mRNA in mouse kidney by in situ hybridization. A, mouse kidney section stained with an 35S-labeled sense CYP2J5 RNA probe showing absence of labeling (negative control). B, adjacent mouse kidney section stained with an 35S-labeled antisense CYP2J5 RNA probe showing intense labeling in the renal cortex and the outer stripe of the outer medulla. C, higher magnification of B showing labeling in both the convoluted and the straight portions of the proximal tubules. D, higher magnification of C showing intense staining throughout the proximal tubules but no labeling in glomeruli or distal tubules. Magnifications × 15 (A and B), 33 (C), and 82 (D).

Regulation of CYP2J5 Expression during Development-- Immunoblotting of microsomal fractions prepared from kidneys of mice ages 18 days fetal to 16 weeks postnatal (adult) with the anti-CYP2J5pep IgG showed that CYP2J5 protein was present before birth, increased gradually during the 1st week of life, reached maximal levels at 2-4 weeks of age, decreased at 8-10 weeks of age, and achieved adult levels by 16 weeks of age (Fig. 7). In contrast, immunoblotting of microsomal fractions prepared from mouse livers with the anti-CYP2J5pep IgG showed that CYP2J5 protein was undetectable prenatally and, during the early postnatal period, first appeared at 1 week of age, remained relatively constant from 1 to 10 weeks of age, and achieved adult levels by 16 weeks of age (Fig. 7). Similar results were obtained using the anti-CYP2J2 IgG that cross-reacts with CYP2J5 (data not shown). Based upon these data, we conclude that the pattern of expression of CYP2J5 during development is different in mouse kidney and liver. Importantly, the age-related changes in CYP2J5 protein expression in mouse kidney correspond to known age-related changes in rodent kidney microsomal arachidonic acid epoxygenase activity (30, 59). Furthermore, maximal CYP2J5 levels occur at a critical time during postnatal kidney development when the pressure-natriuresis relationship is established and when abnormalities in renal function are demonstrable in animals that develop hypertension (60, 61).


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Fig. 7.   Expression of CYP2J5 during development. Microsomal fractions prepared from kidneys and livers of mice ages 18 days fetal to 16 weeks postnatal (adult) were electrophoresed on SDS-10% polyacrylamide gels, transferred to nitrocellulose, and immunoblotted with the anti-CYP2J5pep IgG as described under "Experimental Procedures." Each microsomal sample represents kidney and liver tissues pooled from 3 to 10 animals. The results shown are representative of three separate experiments.

Measurement of Endogenous EETs in Mouse Kidney-- By using a combination of HPLC and GC/MS techniques, we detected substantial amounts of EETs in mouse kidney tissue. Mouse kidney contained ~250 ng of EET/g of kidney. 14,15-, 11,12-, and 8,9-EET were each present in roughly equal amounts (31, 31, and 38% of the total, respectively). The labile 5,6-EET suffers extensive decomposition during the extraction and purification process used and therefore cannot be quantified. Chiral analysis of endogenous mouse kidney EETs showed a slight preference for (14R,15S)- and (11R,12S)-EET (optical purity, 59 and 57%, respectively), whereas 8,9-EET was recovered from mouse kidney as a racemic mixture. The documentation of EETs as endogenous constituents of mouse kidney provides evidence supporting the in vivo metabolism of arachidonic acid by the mouse kidney epoxygenase(s).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

P450-derived eicosanoids have been shown to make important contributions to integrated kidney function either by directly affecting tubular ionic transport processes, vascular tone, and cellular proliferation within the kidney or by mediating the action of several hormones including renin, angiotensin II, and arginine vasopressin (6, 7, 15-26). Furthermore, considerable evidence has accumulated regarding the potential role of these arachidonic acid metabolites in the pathogenesis of hypertension in rodents and humans (6, 7, 27-32). Despite extensive work on the relevance of P450-derived lipid mediators to kidney physiology and pathophysiology, remarkably little is known about the biochemical, molecular, and regulatory properties of the renal P450 enzymes that synthesize these compounds. Herein, we report the cDNA cloning and heterologous expression of a new mouse P450 (CYP2J5) that is abundant in the kidney and active in the biosynthesis of EETs and midchain HETEs. We provide immunologic data to demonstrate that expression of CYP2J5 is regulated during postnatal development, and we present immunohistochemical and in situ hybridization data to show that the CYP2J5 mRNA and CYP2J protein(s) are localized to regions of the nephron where the EETs are known to be biologically active. Finally, we provide GC/MS data to document that the EETs are present endogenously in mouse kidney tissue.

The recombinant CYP2J5 protein catalyzed the NADPH-dependent metabolism of arachidonic acid to 14,15-, 11,12-, and 8,9-EETs, and 11- and 15-HETE; hence, CYP2J5 is both an arachidonic acid epoxygenase and midchain hydroxylase. The CYP2J5 product profile is distinct from that previously reported for other CYP2J enzymes (14, 38, 57, 58). The CYP2J5 product profile is also different from non-CYP2J P450s that are known to metabolize arachidonic acid including members of the CYP1A, CYP2B, CYP2C, CYP2E, and CYP4A subfamilies (5, 12, 33-36, 62-64). Therefore, we conclude that CYP2J5 possesses unique enzymological properties.

The highest concentrations of P450 enzymes in mammalian tissues are found in the liver. As a result, most studies have focused on the molecular and biochemical characterization of hepatic heme-thiolate proteins and their regulatory properties. Despite the fact that the P450 content of kidney microsomes is substantial (20-25% of that in liver) (65), little is known about the renal hemoproteins. Members of the CYP2C and CYP4A subfamilies are constitutively expressed in the rat, rabbit, and human kidney (32-36, 64, 66). In addition, members of the CYP1A, CYP2B, and CYP4A subfamilies are inducible in the rat and rabbit kidney by aromatic hydrocarbons, phenobarbital, and clofibrate, respectively (67, 68). Recent work from our laboratory shows that CYP2J2 is constitutively expressed in human kidney; however, the primary sites of expression of this human hemoprotein are the heart and intestinal tract (38, 41). Work presented in this article confirms our earlier observations that CYP2J enzymes are members of the renal P450 monooxygenase system and shows that a member of the mouse CYP2J subfamily is expressed primarily in the kidney. To our knowledge, this is the first report of a P450 arachidonic acid epoxygenase that is expressed predominantly in mouse kidney.

The CYP4A enzymes, which are active in the omega -terminal hydroxylation of arachidonic acid to 20-HETE, are expressed at highest levels in renal proximal tubules, although CYP4A expression has also been described in renal microvessels (64, 68, 69). The renal distribution of the CYP4A isoforms correlates well with the distribution of renal arachidonic acid omega -hydroxylase activity, which is also highly concentrated in the proximal tubules (69, 70). This suggests that the CYP4A enzymes are the main P450s that biosynthesize 20-HETE within the kidney. In contrast to the localization of 20-HETE formation, epoxygenase activity is distributed throughout the entire nephron with the highest levels of EETs present in proximal tubules and collecting ducts (70). The localization of CYP2J5 mRNA and protein to proximal tubules, together with the demonstration that recombinant CYP2J5 is an arachidonic acid epoxygenase, suggests a role for this enzyme in the biosynthesis of EETs within this portion of the nephron. Interestingly, we also observed CYP2J immunostaining in distal nephron segments, most prominently within the collecting ducts. We are unaware of previous reports documenting expression of a P450 within this region of the nephron. Prostaglandin synthesis is known to occur in collecting ducts, and the renal biological actions of these cyclooxygenase-derived eicosanoids are well described (71). The EETs have been shown to stimulate prostaglandin synthesis in primary cultures of rabbit cortical collecting ducts and to release vasodilator prostaglandins in the isolated-perfused rabbit kidney (19, 25). Thus, CYP2J products could potentially modulate prostaglandin action within distal nephron segments of the kidney.

The cellular distribution of CYP2J enzymes within the kidney may have important functional implications. For example, the localization of CYP2J5 to the proximal tubules suggests that CYP2J5 products may mediate angiotensin II actions, since this portion of the nephron has the highest density of angiotensin II receptors. In this regard, Douglas and co-workers (15, 16) have shown that EETs mediate the angiotensin II-induced rise in cytosolic Ca2+ and mimic the inhibitory effect of angiotensin II on Na+ fluxes from the lumen to the basolateral compartment in the rabbit proximal tubule. Localization of CYP2J protein(s) to the collecting ducts suggests that CYP2J products may be involved in arginine vasopressin action and in ionic transport within this nephron segment. In this regard, P450 epoxygenase metabolites have been shown to inhibit the hydroosmotic effect of arginine vasopressin, decrease net Na+ reabsorption and K+ secretion, and increase cytosolic Ca2+ concentrations in the rabbit cortical collecting tubules (17-19). Importantly, the observed biological effects of EETs within the proximal tubule and cortical collecting ducts occur at concentrations of 10-11 to 10-6 M. These levels are well within the physiologically relevant concentration range based upon estimates of kidney EET concentrations reported herein and described by others (23, 27-29).

Several lines of evidence support the hypothesis that P450-derived arachidonic acid metabolites may be involved in the pathogenesis of hypertension as follows: (a) these eicosanoids have potent effects on vascular tone and fluid/electrolyte transport in the kidney (6, 7, 15-26); (b) the rat renal epoxygenase(s) are under regulatory control by dietary salt, and their inhibition leads to the development of salt-dependent hypertension (27, 28); (c) the salt-sensitive phenotype in the Dahl rat model of genetic hypertension is associated with altered renal metabolism of arachidonic acid (28, 32, 59, 72); (d) alterations in kidney P450 arachidonic acid metabolism exist during the developmental phase of hypertension in spontaneously hypertensive rats (30, 31, 69, 73-75); (e) treatment of spontaneously hypertensive rats with agents that deplete renal P450 normalizes blood pressure (76, 77); and (f) the urinary excretion of epoxygenase metabolites is increased during pregnancy-induced hypertension in humans (29). Recently, the CYP4A genotype has been found to cosegregate with the hypertensive phenotype in Dahl salt-sensitive rats (72). These findings, together with our recent observation that the Cyp2j cluster maps to a region of mouse Chromosome 4 that is close to the mouse Cyp4a cluster (78), suggest that further investigation is warranted to determine if Cyp2j5 is also a viable candidate gene for hypertension.

Many factors are known to alter the levels of expression of hepatic P450s including genetic polymorphism, enzyme induction by xenobiotics and physiological manipulations, and developmental factors. Much less is known about the factors that regulate expression of kidney P450s. Several groups have documented age-related changes in the renal expression of rat CYP4A subfamily P450s (69, 75, 79). The expression of CYP4A1, CYP4A3, and CYP4A8 was maximal between 3 and 5 weeks of age, whereas CYP4A2 increased steadily after 5 weeks of age and was maximal in adult animals (75). None of the CYP4A isoforms were expressed at appreciable levels before 2 weeks of age. In contrast, CYP2J5 was present before birth and reached maximal levels at 2-4 weeks of age. Interestingly, the age-related pattern of CYP2J5 expression was different in kidney and liver, thus suggesting that tissue-specific mechanisms may govern the expression of this hemoprotein during development. Transcriptional activation of liver CYP2E1 during early postnatal development is coincident with specific demethylations in the 5'-end of the CYP2E1 gene which are thought to influence chromatin accessibility to transcription factors (80). Whether similar mechanisms also underlie the regulation of Cyp2j5 gene expression during development are unknown. The developmental pattern of CYP2J5 protein expression in mouse kidney parallels known age-related changes in rodent kidney microsomal arachidonic acid epoxygenase activity (30, 59). Importantly, maximal CYP2J5 protein levels occur during a critical time of postnatal kidney development when the pressure-natriuresis relationship is established and when abnormalities in renal function exist among animals who subsequently develop hypertension (60, 61). Although these temporal relationships are intriguing, they do not prove that the regulation of Cyp2j5 gene expression during this important developmental period impacts on renal function, maintenance of the pressure-natriuresis relationship, or blood pressure.

It is difficult to determine the quantitative contribution of CYP2J5 to renal arachidonic acid metabolism given that specific CYP2J5 inhibitors are not available, the CYP2J antibodies that have been developed are not immunoinhibitory, and CYP2J5 knock-out mice have not yet been generated. However, several lines of evidence support the hypothesis that CYP2J5 plays a major role in renal EET biosynthesis. First, CYP2J5 is present at high levels in mouse kidney. Other mouse P450 epoxygenases, including members of the CYP2C subfamily, appear to be less abundant in the kidney.4 Second, CYP2J5 is localized to regions of the nephron where EETs are known to be produced. Third, age-related changes in CYP2J5 protein expression in mouse kidney correspond to known age-related changes in kidney microsomal arachidonic acid epoxygenase activity. Fourth, CYP2J5 is active in the metabolism of arachidonic acid and has kinetic parameters that are favorable when compared with other renal P450s that metabolize this substrate. For example, CYP2E1 and CYP4A11 have apparent Km values of 228 and 62 µM, respectively (81, 82). Finally, CYP2J5 is more active in the metabolism of arachidonic acid than any other endogenous or xenobiotic substrate we have tested including retinoic acid, testosterone, diclofenac, bufuralol, and various aromatic and heterocyclic amines.5

In summary, we report the cDNA cloning and heterologous expression of CYP2J5, a new mouse P450 that is abundant in the kidney and active in the biosynthesis of EETs and midchain HETEs. We show that this enzyme is regulated during postnatal development and that regulation is tissue-specific. We also demonstrate, using immunohistochemical techniques and in situ hybridization, that CYP2J5 mRNA and CYP2J protein(s) are primarily localized to regions of the nephron where EETs have direct effects on fluid/electrolyte transport and are known to mediate the actions of hormones such as angiotensin II. We conclude that CYP2J5 is an enzymologically distinct, developmentally regulated, heme-thiolate protein that is highly localized to specific nephron segments and contributes to the oxidation of endogenous renal arachidonic acid pools. In light of the well documented effects of epoxyeicosatrienoic acids in modulating renal tubular transport processes, we postulate that CYP2J5 products play important functional roles within the kidney.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Joyce Goldstein for providing purified preparations of CYP2C29, CYP2C37, CYP2C38, CYP2C39, and CYP2C40 and to Dr. Masahiko Negishi for providing CYP2A4 and CYP2A5. We also thank Drs. Richard Philpot and Robert Langenbach for their helpful comments during preparation of this manuscript.

    FOOTNOTES

* These studies were supported in part by National Institutes of Health P01-DK38226 (to M. D. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U62294.

** To whom correspondence should be addressed: Laboratory of Pulmonary Pathobiology, NIEHS, National Institutes of Health, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-1169; Fax: 919-541-4133; E-mail: zeldin{at}niehs.nih.gov.

2 The new sequence that is reported in this paper was submitted to the Committee on Standardized Cytochrome P450 Nomenclature and has been designated CYP2J5.

3 J. Ma and D. C. Zeldin, unpublished observations.

4 D. C. Zeldin and J. A. Goldstein, unpublished observations.

5 C. Crespi, F. Kadlubar, and D. C. Zeldin, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: EET, cis-epoxyeicosatrienoic acid; DHET, vic-dihydroxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid; HPLC, high performance liquid chromatography; P450, cytochrome P450; CYPOR, NADPH-cytochrome P450 oxidoreductase; kb, kilobase; PCR, polymerase chain reaction; GC/MS, gas chromatography/mass spectrometry.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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