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
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ABSTRACT |
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
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INTRODUCTION |
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.
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METHODS |
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.
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RESULTS |
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.
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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.
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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.
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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DISCUSSION |
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.
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.
 |
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