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Am J Physiol Renal Physiol 284: F95-F102, 2003. First published August 27, 2002; doi:10.1152/ajprenal.00132.2002
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Vol. 284, Issue 1, F95-F102, January 2003

Distribution of cytochrome P-450 4A and 4F isoforms along the nephron in mice

David E. Stec1, Averia Flasch2, Richard J. Roman2, and Jared A. White1

1 Department of Physiology and Biophysics, Center for Excellence in Cardiovascular-Renal Research, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505; and 2 Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The production of 20-hydroxyeicosatetraenoic acid (20-HETE) in the kidney is thought to be involved in the control of renal vascular tone and tubular sodium and chloride reabsorption. 20-HETE production in the kidney has been extensively studied in rats and humans and occurs primarily via the actions of P-450 enzymes of the CYP4A and -4F families. Recent advancements in molecular genetics of the mouse have made it possible to disrupt genes in a cell-type-specific fashion. These advances could help in the creation of models that could distinguish between the vascular and tubular actions of 20-HETE. However, isoforms of the CYP4A and -4F families that may be responsible for the production of 20-HETE in the vascular and tubular segments in the kidney of the mouse are presently unknown. The goal of this study was to 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 and glomeruli. In the proximal tubular segments from male mice, the 4A10 and -12 isoforms were present, whereas the 4A10 and -14 isoforms were detected in segments from female mice. In glomeruli, sex differences in the expression pattern of CYP4F isoforms were also observed, with male mice expressing the 4F13, -14, and -15 isoforms, whereas female mice expressed the 4F13, -16, and -18 isoforms. These results demonstrate that isolated nephron and renal vessel segments express multiple isoforms of the CYP4A and -4F families; therefore, elimination of a single CYP4A or -4F isoform may not decrease 20-HETE production in all nephron segments or the renal vasculature of male and female mice. However, the importance of CYP4A vs. -4F isoforms to the production of 20-HETE in each of these renal tubular and vascular segments of the mouse remains to be determined.

cytochome P-450 4A isoforms; cytochrome P-450 4F isoforms; nephron segment; mouse; microdissection; reverse transcription-polymerase chain reaction; 20-hydroxyeicosatetraenoic acid


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

20-HYDROXYEICOSATETRAENOIC ACID (20-HETE) is the major metabolite of arachidonic acid (AA) produced in the kidney of several species, including humans (22). 20-HETE has been reported to regulate renal vascular tone (12, 30), mediate tubuloglomerular feedback (31), and inhibit electrolyte transport in several nephron segments (6, 20). Alterations in renal 20-HETE production may also contribute to the development of hypertension in both the spontaneously hypertensive rat (SHR) and the Dahl salt-sensitive (Dahl S) rat (21, 24, 25).

Three isoforms of the CYP4A family have been cloned in the mouse and 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, with levels in males being much higher than those in females. The CYP4A10 and CYP4A14 isoforms are expressed at lower levels in the mouse kidney under basal conditions; however, both isoforms were markedly inducible by treatment with the peroxisome proliferator methylclofenapate (2, 8). Although mouse CYP4A isoforms share a high degree of amino acid identity with rat CYP4A isoforms (7), the catalytic activity of mouse CYP4A10 and CYP4A12 isoforms in regard to the production of 20-HETE has yet to be determined. In the human kidney, the only isoform of the CYP4A family to be cloned is CYP4A11 (2). However, in contrast to the rat, recent studies have indicated that the primary isoforms responsible for the production of 20-HETE in the human kidney are members of the CYP4F family (4, 16).

In mice, five members of the CYP4F family have also been identified. The CYP4F15 and -16 isoforms have been demonstrated to be expressed in the kidney under basal conditions, with the CYP4F16 isoform being the more abundant transcript (5). In the kidney, the CYP4F15 isoform is highly inducible by clofibrate, whereas clofibrate has no effect on CYP4F16 levels (5). Sex differences in the levels of CYP4F expression in the kidney have yet to be reported. Although CYP4F isoforms in the human kidney have been demonstrated to produce 20-HETE (4, 16), the ability of the CYP4F isoforms expressed in the kidneys of mice and rats to metabolize AA and produce 20-HETE has not yet been demonstrated.

The dissection of complex paracrine systems has been successfully accomplished by either targeted overexpression or disruption of specific genes in transgenic and gene knockout mice. Advances in mouse genetics allow for targeted overexpression and deletion of genes in specific nephron segments of the kidney (17, 26). The development of these approaches would greatly increase our understanding of the physiological consequence of altered renal tubular or vascular 20-HETE production. Targeted disruption of the CYP4A14 gene has been described (10); however, instead of decreasing renal 20-HETE production, loss of this isoform was associated with increased production of 20-HETE in the kidney and hypertension in male mice. The increased 20-HETE production in these mice was attributed to an increase in the expression of the CYP4A12 isoform; however, changes in the expression of CYP4F isoforms were not considered in the previous study (10). The results from that study indicate that 20-HETE produced in the kidney of the mouse may be derived from multiple isoforms of the CYP4A and CYP4F families in a sex- and cell-type-specific fashion. In the present study, we set out to identify the isoforms of the CYP4A and CYP4F families present in specific nephron segments and renal vessels of the mouse. This was accomplished by designing specific primers to amplify the individual isoforms of the CYP4A and CYP4F families and performing RT-PCR on bulk isolated nephron segments and renal microvessels from both male and female mice.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Studies were performed in 16- to 24-wk old male and female C57BL/6J mice purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were housed under standard conditions and allowed full access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Mississippi Medical Center.

Isolation of proximal tubules, thick ascending limb of the loop of Henle, and collecting ducts. Mice were anesthetized with pentobarbital sodium (50 mg/kg), and the abdominal aorta was cannulated below the kidneys. The mesenteric and celiac arteries were tied off to increase delivery of solutions to the kidneys. The kidneys were flushed through the aorta with 3 ml of cold wash solution consisting of (in mM) 135 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 2 KH2PO4, 5.5 glucose, 5 L-alanine, and 10 HEPES (pH 7.4). The kidneys were immediately removed, and the cortex and outer medulla were separated under a dissecting microscope. The cortex was then sectioned into 1-mm-thick coronal sections using a Stadie-Riggs microtome, and the outer medulla was finely minced with scissors. Cortical sections as well as the outer medulla were then incubated separately in dissection solution at 37°C for 1 h while O2 was continuously blown over the mix. The dissection solution consisted of wash solution with the following additives: 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 soybean trypsin inhibitor (Sigma). Proximal tubules were then isolated by separation on a Percoll gradient (28), and individual tubule segments (30 mm in total length) were collected under a stereomicroscope and placed in 100 µl of lysis buffer for RT-PCR-specific RNA isolation (RNAqueous, Ambion, Austin, TX).

Thick ascending limbs of the loop of Henle (TALH) were isolated from the outer medulla of the kidney after enzymatic digestion, and the supernatant was collected and placed on a 70-µm nylon sieve. The sieve was rinsed several times with ice-cold digestion solution, and the retained tissue was washed off the sieve with a wash solution containing 1% BSA. Individual TALH segments were then collected under a stereomicroscope with fine forceps and placed into the lysis solution for RNA isolation. Collecting ducts were isolated from 1-mm-thick coronal sections of the whole kidney after a 1-h incubation in digestion solution at 37°C while O2 was continuously blown over the mix. After the digestion period, both cortical and medullary collecting ducts were collected under a stereomicroscope with fine forceps and placed into the lysis solution for RNA isolation.

Isolation of renal microvessels and glomeruli. Renal vessels were isolated from the kidney using a modified Evans blue technique (13). Briefly, the kidney was first flushed with 3 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 homogenized several times with a Polytron homogenizer. The resulting suspension was then passed several times first through a 20-gauge needle, and then through a 22-gauge needle. The suspension was then passed though a 180-µm mesh sieve that was atop a 100-µm mesh sieve. After several washes in wash solution with 1% BSA, the material retained by each of the sieves was collected. Each microvascular fraction was examined under a stereomicroscope, and most vessel segments were found to be totally devoid of glomeruli and tubules. Clean vessel segments were then collected using fine forceps and placed into the lysis solution for RNA isolation. Glomeruli were isolated by a rapid sieving technique as previously described (23). The material passing through the 180-µm sieve after the kidney tissue was forced through was collected and passed though a 100-µm sieve and then through a 70-µm sieve. The 70-µm sieve was rinsed several times in wash solution with 1% BSA, and the material was collected. The fraction was then examined under a stereomicroscope, and individual glomeruli devoid of visible tubular contamination were collected (100 total) and place into lysis solution for RNA isolation.

RNA isolation and RT-PCR. RNA was isolated using a commercially available kit specifically designed for RT-PCR according to the manufacturer's guidelines (RNAqueous, Ambion). RNA was eluted in a 60-µl volume with nuclease-free water. Ten microliters of RNA were then used in a reverse transcription reaction using an oligo-dT primer with avian myeloblastosis virus (AMV)-RT in a 20-µl final volume. To control for amplification of genomic DNA, reactions lacking AMV-RT were run in parallel. PCR was performed on 1 µl of the reverse transcription reaction in a 25-µl volume using standard PCR reaction conditions. The PCR reactions for the CYP4A isoforms were cycled as follows: 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 CYP4F isoforms were identical, with the exception of a 55°C annealing temperature. PCR primers for the CYP4A isoforms were as follows: CYP4A10 (GenBank accession no. AB018421), sense 5'-GACAAGGACCTACGTGCTGAGG, antisense 5'-CTCATAGCAAATTGTTTCCCA; CYP4A12 (Y10222), sense 5'-TGAGTCCTATGAAAGAGTGCC, antisense 5'-CTGGAAGCCCAGCAGAAGGTG; and CYP4A14 (Y11640), sense 5'-CCCACAGGGACATGCAGATTAG, antisense 5'-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, antisense CYP4F13- 5'-AGGAGGCAGTTCTGTTTATTCA; CYP4F14 (AF233644), sense 5'-AGCTCACCTCTGGCATTTATTCC, antisense 5'-CTCAGACATCCCTTTGGCTTCCTA; CYP4F15 (AF233645), sense 5'-TCCGCTTTGACCCAGAGAATA, antisense 5'-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. The primers amplified a 405-bp fragment. PCR products were separated on 0.8% 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 from all isoforms amplified from either whole kidney or liver were cloned into TA cloning vectors (Invitrogen, Carlsbad, CA). Two independent clones for each isoform were then sequenced to confirm specificity of PCR primers.

Measurement of AA metabolism. Mice were anesthetized with pentobarbital sodium (50 mg/kg), and the kidneys were flushed with an ice-cold washing solution as noted above. The kidneys were then rapidly removed and sectioned into cortex and outer medulla. Outer medullas from two mice were pooled to obtain sufficient amounts of tissue for the microsome preparation. Microsomes from the liver were also prepared. The tissue was homogenized in 3 ml potassium phosphate buffer containing (in mM) 10 potassium phosphate, 25 sucrose, 1 EDTA, and 0.1 PMSF. Microsomes were prepared by differential centrifugation and resuspended in a buffer that contained (in mM) 100 potassium phosphate, pH 7.2, 1 dithiothreitol, 1 EDTA, and 0.1 PMSF as well as 30% glycerol.

Enzyme activity was measured by incubating microsomes from the cortex and outer medulla of the kidney or the liver (0.5 mg protein) with a saturating concentration of [14C]AA (1 µCi; 42 µM) in an NADPH-regenerating system as previously described (24). Additional experiments were then performed comparing the ability of microsomes prepared from the kidneys of male and female rats to metabolize AA using a lower concentration of AA (1 µCi; 1.8 µM), because the percent conversion of AA to 20-HETE in renal microsomes of C57/BL6 mice was found to be very low relative to rats, rabbits, and other species as reported previously (1). The reactions were terminated by acidification with formic acid, extractions were made twice with ethyl acetate, and the preparation was dried under N2 gas. The metabolites were then resuspended in 500 µl of 100% ethanol and separated by HPLC using a C18 reverse-phase column (2.1 × 250 mm, 5 µm; Supelco) and a 2-cm guard column. A linear elution gradient ranging from acetonitrile-water-acetic acid (50:50:2 vol/vol/vol) to acetonitrile-acetic acid (100:0.2 vol/vol) was used. Metabolites were monitored by a radioactive flow detector modified with lead shielding for a low background. The mean production rate for each metabolite was calculated and expressed as picomoles formed per minute per milligram of protein.

Statistics. Values are presented as means ± SE. The significant difference in mean values was evaluated by an unpaired t-test. A value of P < 0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of CYP4A isoforms in nephron segments and renal vessels. In the proximal tubule, the CYP4A10 and CYP4A12 isoforms were the only isoforms detected in male mice (Fig. 1A). This was in contrast to female mice, in which the CYP4A10 and CYP4A14 isoforms were detected in the proximal tubule (Fig. 1B). The CYP4A12 isoform could not be amplified from proximal tubules isolated from female mice. The CYP4A10 isoform was the only isoform that could be amplified from the TALH and collecting ducts of both male and female mice (Figs. 2 and 3). In the renal vessels, the CYP4A10 isoform was the only isoform that could be detected in both large (>180-µm diameter) and small (between 180- and 100-µm diameter) vessels (Fig. 4). There were no sex differences in the expression pattern of CYP4A isoforms in the renal vasculature (Figs. 4 and 5). The CYP4A10 isoform was also the only isoform expressed in isolated glomeruli from both male and female mice (Fig. 6).


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  		Fig. 1.   RT-PCR distribution of CYP4A and CYP4F isoforms in isolated proximal tubule segments of male (A) and female (B) mice. The CYP4A isoforms detected in the male were CYP4A10 and CYP4A12, whereas the CYP4A10 and CYP4A14 isoforms were detected in females. The pattern of expression of the CYP4F isoforms was 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 thick ascending loop of Henle (TALH) segments of male (A) and female (B) mice. Only the CYP4A10 isoform was detected in both male and female mice. The CYP4F13 and CYP4F16 isoforms were both detected in isolated TALH segments from male and female mice.



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  		Fig. 3.   Distribution of CYP4A and CYP4F isoforms in isolated cortical and medullary collecting duct segments of male (A) and female (B) mice by RT-PCR. The CYP4A10, CYP4F13, and CYP4F16 isoforms were detected in both male and female mice.



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  		Fig. 4.   RT-PCR analysis of CYP4A and CYP4F isoforms from large (>180-µm) renal vessels from male (A) and female (B) mice. The CYP4A10, CYP4F13, CYP4F16, and CYP4F18 isoforms were all expressed in large renal vessel segments from both male and female mice.



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  		Fig. 5.   RT-PCR distribution of CYP4A and CYP4F isoforms from small renal vessels from male (A) and female (B) mice. Vessels were <180 µm but >100 µm in diameter. The CYP4A10 and CYP4F13 isoforms were the only isoforms detected in both male and female mice.



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  		Fig. 6.   Distribution of CYP4A and CYP4F isoforms in isolated glomeruli from male (A) and female (B) mice as determined by RT-PCR. The CYP4A10 isoform was detected in glomeruli from both male and female mice. The CYP4F13, CYP4F14, and CYP4F15 isoforms were all present in glomeruli isolated from male mice. In glomeruli from female mice, the CYP4F13, CYP4F16, and CYP4F18 isoforms were all detected.

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


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  		Fig. 7.   Summary of the distribution of CYP4A and CYP4F isoforms in renal microvessels and tubule segments in mice. Blue bars, male mice; orange bars, female mice; LV, large renal vessels (>180-µm diameter); SV, small renal vessels (between 180- and 100-µm diameter); Glom, glomeruli; PT, proximal tubule; TLH, thin decending and ascending loops of Henle; TALH, thick ascending loop of Henle; CD, collecting ducts.

AA metabolism. We first characterized the renal metabolism of AA in the kidney and liver 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 in Table 1. Microsomes prepared from the livers of these mice avidly produced 20-HETE; 14,15-, 11,12-, 8,9-, and 5,6-diHETEs; 18-, 19-, 16- 15-, 12-, 5-, and other HETEs; and lesser quanities of epoxyeicosatrienoic acids (EETs). The overall production rate of 20-HETE was similar to the levels previously reported in rat liver microsomes (14). Mouse renal cortical microsomes produced primarily 20-HETE, with lesser quantities of EETs and diHETEs, and largely 15- and 12-HETEs. However, the catalytic activity of the microsomes to produce 20-HETE and EETs was about five times lower than that seen in the livers of these same mice or comparable to the levels previously observed in renal cortical microsomes in rats (1, 14). We tried a number of different substrate concentrations, cofactor additions, incubation times, and amounts of protein (0.25-2 mg) in the reactions but could not increase conversion rates.

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

Because of the low conversion rates of AA to EETs and 20-HETE, we therefore chose to compare the relative activity of microsomes prepared from the kidneys of male and female rats using only [14C]AA (1.8 µM) to maximize the conversion rates and our ability to detect differences in activity. The production of 20-HETE was lower in microsomes prepared from the renal cortices of female mice compared with male mice (1.38 ± 0.13 vs. 1.71 ± 0.12 pmol · min-1 · mg protein-1); however, this difference did not reach statistical significance (Table 2). The production of 20-HETE in the outer medulla of male and female mice was not statistically different. Epoxygenase activity was significantly lower in microsomes prepared from the cortex and outer medulla of female mice compared with male mice, with the cortical values averaging 2.90 ± 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/6J mice


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The goal of the present study was to examine the distribution of CYP4A and CYP4F isoforms along the nephron and in renal microvessels of C57BL/6J mice to determine what 20-HETE-producing enzymes may be present in these tubular and vascular segments. This information is especially useful in the context of defining an experimental approach to reduce the production of 20-HETE in one of these segments by elimination of a CYP4A or CYP4F isoform. Although the CYP4A10 and CYP4F13 isoforms were detected in every tubular and vascular segment examined, some segments also express other CYP4A and CYP4F isoforms that may be able to produce 20-HETE if the CYP4A10 or CYP4F13 isoforms were inactivated by gene targeting. For example, in the TALH of mice the CYP4A10 isoform is present as well as two isoforms of the CYP4F family. Elimination of this CYP4A isoform would leave the other CYP4F isoforms still intact, which might be sufficient to produce 20-HETE in this nephron segment. Previous studies have demonstrated that 20-HETE is a regulator of chloride transport in the TALH (6) and that decreased production of 20-HETE in the TALH may be responsible for the shifting of the pressure-naturetic response and the development of hypertension in Dahl S rats (15). However, this hypothesis may be difficult to test in mice in which the CYP4A10 isoform has been deleted in the TALH because this nephron segment expresses multiple CYP4F isoforms. Similarly, 20-HETE has been demonstrated to be a potent vasoconstrictor of the renal microcirculation (12), and alteration in the production of 20-HETE in renal arterioles is thought to contribute to the development of hypertension in the SHR (25, 29). Even though the CYP4A10 isoform was the only isoform detected in renal blood vessels of male and female mice, disruption of this isoform may not lead to a decrease in 20-HETE production in renal blood vessels due to the presence of the CYP4F13 isoform in small vessels and multiple CYP4F isoforms in large vessels.

In the present study, we observed numerous sex differences in the distribution of CYP4A and CYP4F isoforms in different tubular and vascular segments. In the proximal tubule, male mice expressed the CYP4A10 and CYP4A12 isforms, while the CYP4A10 and CYP4A14 isoforms were detected in females. The expression of the CYP4A14 isoform, but not the CYP4A12 isoform, in female mice under basal conditions is in agreement with previous studies using Northern blot analysis of whole kidney RNA (10). However, castration of male mice leads to a marked increase in the levels of the CYP4A14 and decrease in the CYP4A12 isoform in the kidney, suggesting a role for testosterone as a negative and positive regulator of these genes in mice. Both of these changes in expression can be prevented by treatment of mice with dihydrotestosterone (DHT) (10). Interestingly, renal 20-HETE production is correlated with changes in the expression of the CYP4A12 isoform after castration and DHT treatment (10), suggesting that this isoform may be responsible for the higher rate of 20-HETE production in the renal cortex of male vs. female mice. Previous studies have also detected increased levels of lauric acid hydroxylase activity and CYP4A protein in the kidneys of male vs. female mice (9).

The only sex difference in distribution of the CYP4F isoforms was found in the glomerulus. Glomeruli from male mice expressed the CYP4F13, CYP4F14, and CYP4F15 isoforms, whereas those from female mice expressed the CYP4F13, CYP4F16, and CYP4F18 isoforms. Expression of the CYP4F15 isoform in the glomeruli of male mice was the only segment in which the CYP4F15 isoform was detected in the present study. Interestingly, this isoform could not be amplified from whole kidney RNA samples but could be readily amplified from the liver (Stec, unpublished observations). If the CYP4F15 isoform avidly metabolizes AA to 20-HETE, the differential expression of CYP4F15 in the liver vs. the kidney may contribute to the present observation that the production of 20-HETE is much greater in the liver vs. the kidney of C57BL/6J mice. This concept is consistent with the results of recent studies indicating that the CYP4F2 is the primary isoform responsible for the production of 20-HETE in the human kidney (4, 16), whereas other human isoforms such as CYP4F8 and CYP4F12 appear to primarily metabolize prostaglandins and leukotrienes (3, 19). The murine CYP4F14, -15, -16, and -18 isoforms share a high degree of amino acid 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 CYP4F isoforms to metabolize AA vs. other substrates is unknown, so it is difficult to speculate on which isoform is most important in producing 20-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

To determine the functional significance of the observed differences in CYP4A and CYP4F isoform distribution in male vs. female C57BL/6J mice, we measured the metabolism of AA by microsomes prepared from the cortex and outer medulla of the kidney. Despite the fact that we characterized clear sex differences in the expression of CYP4A and CYP4F mRNA in various nephron segments of the kidney, we did not detect any significant difference in the production of 20-HETE by microsomes prepared from the renal cortex or outer medulla of male and female C57BL/6J mice. The reason for this observation remains to be determined, but there are many possible explanations. For example, CYP4A and CYP4F proteins in the kidney may be different from the isoforms detected by RT-PCR in the present study. It is also possible that many of the murine isoforms do not metabolize AA to 20-HETE but are primarily involved in the hydroxylation of prostaglandins and leukotrienes in vivo.

Interestingly, we found that the production of 20-HETE was much lower in the kidneys of mice compared with that reported in other species and about five times lower than that seen in the livers of the same animals (1, 14). The low level of 20-HETE production in the kidneys of C57BL/6J female mice is in agreement with previous reports of wild-type CYP4A14 knockout mice on a 129 mixed genetic background (10). Previous reports of renal 20-HETE production in male mice have indicated higher levels in both a mixed 129 genetic background (10) as well as in NMRI mice (11). Because we only examined distribution of the CYP4A and CYP4F isoforms in C57BL/6J mice in the present study, it is possible that these observed discrepancies in 20-HETE production may be due to differences in the distribution of these isoforms between various inbred strains. The low level of metabolism of AA by renal microsomes from C57BL/6J mice may indicate that AA is not the preferred substrate for the CYP4A and CYP4F isoforms in vivo. This hypothesis is supported by previous studies that have demonstrated much higher levels of lauric acid hydroxylation by renal microsomes compared with AA hydroxylation in other inbred strains of mice (9, 11, 27). Also, studies with purified CYP4A14 protein indicate that this isoform is not able to metabolize AA but is able to efficiently metabolize lauric acid (10). The CYP4A10 isoform shares 92% amino acid homology with the rat CYP4A1 isoform, whereas the CYP4A12 and CYP4A14 isoforms share the greatest homology with rat CYP4A8 and CYP4A3 isoforms, respectively (Table 2). The rat CYP4A1 isoform has been demonstrated to have 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 of homology with the rat CYP4A isoforms, the specific ability of the mouse CYP4A10 and CYP4A12 isoforms to generate 20-HETE has yet to be reported.

The mouse provides a powerful tool by which the various physiological actions of 20-HETE in the kidney can be cleanly dissected in vivo. We have identified the isoforms of the CYP4A and CYP4F families throughout the nephron and in renal vessels that may be responsible for the production of 20-HETE in vivo. Several differences in the distribution of CYP4A and CYP4F isoforms were identified in specific renal tubular and vascular segments analyzed in male and female C57BL/6J mice. Disparities in the CYP4A and CYP4F isoforms may account for the altered levels of renal 20-HETE production between various inbred strains of mice as well as the difference in 20-HETE production between male and female mice. The specific substrate specificities of each of the murine CYP4A and CYP4F isoforms need to be determined to fully test this hypothesis. These strain differences in renal 20-HETE production need to be taken into account when the appropriate genetic background in which to alter the levels of renal 20-HETE production in mice is being considered. Strains such as the C57BL/6J exhibit low levels of renal 20-HETE production and would not be an ideal genetic background for models to examine reduced renal 20-HETE production by gene knockout; however, this strain may be appropriate when the physiological effects of increased renal 20-HETE production via cell-type-specific transgenic targeting are being examined. These types of transgenic and gene-targeted models will be critical to fully determine the complex role of intrarenal 20-HETE production in the regulation of blood pressure and renal function.


    ACKNOWLEDGEMENTS

This work was supported by an American Heart Association, Heartland Affiliate, Beginning-Grant-in-Aid (to D. E. Stec) and National Heart, Lung, and Blood Institute Grant HL-51971.


    FOOTNOTES

Address for reprint requests and other correspondence: D. E. Stec, Dept. of Physiology and Biophysics, Ctr. for Excellence in Cardiovascular-Renal Research, 2500 North State St., Jackson, MS 39216-4505 (E-mail: dstec{at}physiology.umsmed.edu).

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.

September 3, 2002;10.1152/ajprenal.00132.2002

Received 9 April 2002; accepted in final form 26 August 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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