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1General Clinical Research Center, University of North Carolina School of Medicine, Chapel Hill 27514; 2Duke University School of Medicine, Durham, North Carolina 27705; 3Department of Pharmaceutics, University of Washington, Seattle, Washington 98195; and 4Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38105
Submitted 31 March 2003 ; accepted in final form 15 May 2003
ABSTRACT
A single-nucleotide polymorphism (A6986G) in the cytochrome P-450 3A5 (CYP3A5) gene distinguishes an expressor (*1) and a reduced-expressor (*3) allele and largely predicts CYP3A5 content in liver and intestine. CYP3A5 is the prevailing CYP3A isoform in kidney. We report that, among renal microsomes from 21 organ donors, those from *1/*3 individuals had at least eightfold higher mean kidney microsomal CYP3A5 content and 18-fold higher mean CYP3A catalytic activity than did those from *3/*3 individuals (P = 0.0001 and P = 0.0137, respectively). We also report significant associations between the A6986G polymorphism and systolic blood pressure (P = 0.0007), mean arterial pressure (P = 0.0075), and creatinine clearance (P = 0.0035) among 25 healthy African-American adults. These associations remained significant when sex, age, and body mass index were taken into account. The mean systolic blood pressure of homozygous CYP3A5 expressors (*1/*1) exceeded that of homozygous nonexpressors (*3/*3) by 19.3 mmHg. We speculate whether a high CYP3A5 expressor allele frequency among African-Americans may contribute to a high prevalence of sodium-sensitive hypertension in this population.
cytochrome P-450 3A5; single-nucleotide polymorphism; kidney; sodium sensitivity
-hydroxycortisol, a marker of in vivo
CYP3A activity (16).
A role for CYP3A enzymes in BP control is scientifically plausible.
6-Hydroxysteroid products of CYP3A-catalyzed reactions stimulate sodium
transport across A6 toad kidney cells and sodium retention in rats
(1,
3,
11). CYP3A-mediated
6-hydroxylation is a major metabolic pathway for glucocorticoids and
mineralocorticoids, including aldosterone, and previous evidence suggests
CYP3A modulation of A6 cell sodium transport through a mineralocorticoid
receptor mediated mechanism
(13). Consistent with data
from rat kidney (1),
immunostaining for CYP3A highlights human distal tubules and collecting ducts
(14), the principal sites at
which mineralocorticoids stimulate sodium reabsorption. Renal CYP3A-produced
6-hydroxysteroids have been postulated to function as mineralocorticoids
near their site of synthesis
(1), and CYP3A activity, along
with that of 11-hydroxysteroid dehydrogenase type 2, has been proposed
to regulate glucocorticoid occupancy of mineralocorticoid receptors
(13).
The lack of previous reports associating human renal CYP3A and BP may partly reflect the previous unavailability of noninvasive methods to predict renal CYP3A activity. It is now established that, among CYP3A isoforms, CYP3A5 expression prevails in human kidney (5). Recent reports demonstrate that CYP3A5 expression in liver (9) and intestine (10) is polymorphic and largely determined by a single-nucleotide polymorphism (A6986G) that distinguishes the CYP3A5*1 ("expressor") allele from the *3 (a "reduced-expressor") allele. We demonstrate that CYP3A5 genotype similarly predicts renal CYP3A5 expression. We also report preliminary evidence of CYP3A5 genotype prediction of BP and creatinine clearance (CrCl) among a group of healthy young adults.
METHODS
We assayed CYP3A catalytic activity and CYP3A5 immunoreactivity in renal tissue samples from 21 organ donors: 17 were Caucasian, 1 was Hispanic, and 3 were of unknown ethnicity. All methods were described previously (10). CYP3A5 genotypes were determined by direct genomic DNA sequencing. Microsomes were isolated by differential centrifugation. CYP3A5 content in 50 µg of microsomes was measured by Western blot with a CYP3A5-specific antibody (BD Gentest, Woburn, MA); undetectable bands were assigned a value of 0.25 pmol/mg, the limit of quantitation (LOQ; Fig. 1A). Midazolam (MDZ) 1'-hydroxylation was measured after a 15-min incubation of 200 µg protein with 8 µM MDZ; microsomes with undetectable activity were given the LOQ value of 0.04 pmol · min-1 · mg-1.
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We genotyped 89 unrelated volunteers for the CYP3A5 A6986G single-nucleotide polymorphism using previously described methods (9); all subjects were self-identified African-Americans. Among the subset of this group consisting of 25 healthy individuals (age range: 18-52 yr) who volunteered to be screened for a pharmacogenetic study, we tested the ancillary hypothesis of CYP3A5 genotype association with BP and CrCl. Nurses blind to subject genotypes and medical histories performed and recorded all clinical measurements. Studies were approved by the University of North Carolina Committee on the Protection of Human Research Subjects.
RESULTS
The average CYP3A5 content (1.98 pmol/mg, n = 5) of microsomes from *1/*3 kidneys exceeded the average of those from *3/*3 kidneys (n = 16, Wilcoxon P = 0.0001), which was at or below the LOQ (Fig. 1A). None of the kidneys was *1/*1. The closely related CYP3A4 was not detected with a CYP3A4-specific antibody in any of the microsomal preparations. Mean microsomal CYP3A activity, reflected by MDZ 1'-hydroxylation, was 18-fold higher in *1/*3 kidney microsomes (8.04 pmol · min-1 · mg-1) than in those from *3/*3 kidneys (0.43 pmol · min-1 · mg-1, Wilcoxon P = 0.0137; Fig. 1B). Analysis of DNA from the *1/*3 outlier, indicated by arrows in Fig. 1, revealed an inactivating frame-shift mutation (27131-27132insT) that produces an inactivating allele known as *7 (6). Removal of this outlier slightly lowered P values.
CYP3A5*1 allele frequency among 89 African-Americans was 0.7.
Among the 25-individual subset (Table
1), CYP3A5 genotype associated with seated systolic BP
(SBP), mean arterial pressure, the product of SBP and heart rate (HR) (SBP *
HR, an indicator of left-ventricular oxygen consumption), and Cockcroft-Gault
CrCl. The *1/*1 group averaged the highest value for each
measure. Average *1/*1 SBP exceeded that of the
*3/*3 group by 19.3 mmHg
(Fig. 2), and a gene-dose
effect was apparent. CYP3A5 genotype associated significantly with
combined BP strata (P = 0.0048;
Table 1) from the Sixth Report
of the Joint National Committee on Prevention, Detection, Evaluation, and
Treatment of High BP {N = optimal or normal [SBP <130 and diastolic BP
(DBP) <85 mmHg]; H = high-normal or higher (SBP 
130 or DBP 85
mmHg)} (7).
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Sex-specific analysis detected CYP3A5 genotype associations with SBP, mean arterial pressure, and estimated CrCl among females (Table 1); similar trends were noted among the smaller sample of males, but these did not reach statistical significance. In multiple regression analyses, sex and genotype accounted for 70% of the variability in SBP. Compared with non-*1/*1 females, *1/*1 males showed a >20-fold increased risk of high normal or higher BP (P = 0.0002).
Subject age, averaging 
25 yr, did not vary between genotype groups.
Age and body mass index effects on BP were nonsignificant. Genotype
association with body mass index, DBP, pulse pressure, and HR did not reach
statistical significance.
DISCUSSION
CYP3A5*1 allele associates with CYP3A5 expression in human kidney, as previously reported for liver and intestine. However, in the liver and intestine, there is also substantial CYP3A4 expression (9, 10). As a result, hepatic and intestinal aggregate CYP3A activities associate only weakly with the CYP3A5*1 allele. In contrast, CYP3A4 was not detected in renal microsomes, and mean CYP3A activity differed markedly between CYP3A5 genotype groups.
Our preliminary finding of CYP3A5 genotype association with resting BP among healthy adults may be consistent with a role for CYP3A enzymes in BP control. Young adults with supernormal BP have an increased long-term risk of death due to cardiovascular and coronary heart disease (12); the identification of common genetic polymorphisms relevant to BP control is thus an important line of investigation. We are presently testing our hypothesis of a CYP3A5 genotype-BP correlation in a larger sample of adults. If shown true, our conjecture might portend a role for CYP3A5 inhibition in the treatment of some forms of hypertension.
The CYP3A5*1 allele frequency among our cohort of African-Americans agrees roughly with a previous report (6). This frequency exceeds those among all other ethnic populations studied to date (6, 9). Interethnic differences in the prevalence of sodium sensitivity (17) parallel those of hypertension (2), with African-Americans having the highest global prevalence of each. A possible link between CYP3A5 activity and the high prevalence of sodium-sensitive hypertension among African-Americans may merit further study.
DISCLOSURES
This study was supported by the Doris Duke Medical Research Program and National Institutes of Health Grants RR-00046, GM-37149, GM-63666, and GM-60346.
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FOOTNOTES |
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FOOTNOTES
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.
REFERENCES
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