Overexpression of cytochrome P450 4F2 in mice increases 20-hydroxyeicosatetraenoic acid production and arterial blood pressure
2009; Elsevier BV; Volume: 75; Issue: 12 Linguagem: Inglês
10.1038/ki.2009.67
ISSN1523-1755
AutoresXiaoliang Liu, Yanyan Zhao, Wang Lu-zeng, Xianghong Yang, Zhihong Zheng, Yuanyuan Zhang, Fangjie Chen, Hong Liu,
Tópico(s)Alcohol Consumption and Health Effects
ResumoCytochrome P450 4F2 (CYP4F2) activity is thought to be a factor in the pathogenesis of hypertension through its bioactive metabolite 20-hydroxyeicosatetraenoic acid (20-HETE). We previously found that a gain-in-function CYP4F2 variant in a Chinese cohort was associated with elevated urinary 20-HETE and hypertension. To further explore this association we generated a transgenic mouse model expressing CYP4F2 driven by a modified mouse kidney androgen-regulated protein promoter. This heterologous promoter regulated the expression of luciferase and his-tagged CYP4F2 in transfected HEK 293 cells. In the kidney of transgenic mice, CYP4F2 was localized to renal proximal tubule epithelia and was expressed at a higher level than in control mice, leading to increased urinary 20-HETE excretion. Assessment of CYP4F2 activity by an arachidonic acid hydroxylation assay showed that 20-HETE production was significantly higher in kidney microsomes of transgenic mice compared to control mice, as was their systolic blood pressure. There was a positive correlation of blood pressure with urinary 20-HETE levels. Our results show that increased expression of CYP4F2 in mice enhanced 20-HETE production and elevated blood pressure. Cytochrome P450 4F2 (CYP4F2) activity is thought to be a factor in the pathogenesis of hypertension through its bioactive metabolite 20-hydroxyeicosatetraenoic acid (20-HETE). We previously found that a gain-in-function CYP4F2 variant in a Chinese cohort was associated with elevated urinary 20-HETE and hypertension. To further explore this association we generated a transgenic mouse model expressing CYP4F2 driven by a modified mouse kidney androgen-regulated protein promoter. This heterologous promoter regulated the expression of luciferase and his-tagged CYP4F2 in transfected HEK 293 cells. In the kidney of transgenic mice, CYP4F2 was localized to renal proximal tubule epithelia and was expressed at a higher level than in control mice, leading to increased urinary 20-HETE excretion. Assessment of CYP4F2 activity by an arachidonic acid hydroxylation assay showed that 20-HETE production was significantly higher in kidney microsomes of transgenic mice compared to control mice, as was their systolic blood pressure. There was a positive correlation of blood pressure with urinary 20-HETE levels. Our results show that increased expression of CYP4F2 in mice enhanced 20-HETE production and elevated blood pressure. The human cytochrome P450 4F2 (CYP4F2) gene (MIM no. 604426) encodes an ω-hydroxylase that catalyzes the arachidonic acid (AA) to 20-hydroxyeicosatetraenoic acid (20-HETE).1.Lasker J.M. Chen W.B. Wolf I. et al.Formation of 20-hydroxyeicosatetraenoic acid, a vasoactive and natriuretic eicosanoid, in human kidney. Role of CYP4F2 and CYP4A11.J. Biol Chem. 2000; 275: 4118-4126Crossref PubMed Scopus (267) Google Scholar Previous studies have demonstrated that 20-HETE participates in the development of hypertension by regulating vascular and renal tubular functions.2.McGiff J.C. Quilley J. 20-HETE and the kidney: resolution of old problems and new beginnings.Am J Physiol. 1999; 277: R607-R623Crossref PubMed Google Scholar, 3.Miyata N. Roman R.J. Role of 20-hydroxyeicosatetraenoic acid (20-HETE) in vascular system.J Smooth Muscle Res. 2005; 41: 175-193Crossref PubMed Scopus (194) Google Scholar, 4.Roman R.J. P-450 metabolites of arachidonic acid in the control of cardiovascular function.Physiol Rev. 2002; 82: 131-185Crossref PubMed Scopus (1169) Google Scholar, 5.Sarkis A. Roman R.J. Role of cytochrome P450 metabolites of arachidonic acid in hypertension.Curr Drug Metab. 2004; 5: 245-256Crossref PubMed Scopus (73) Google Scholar On one hand, 20-HETE increases peripheral vascular resistance by potently constricting small arteries or by sensitizing the vascular smooth muscle cells to constrictor and myogenic stimuli. On the other hand, 20-HETE causes natriuresis by inhibiting sodium reabsorption. Recently, we found that a functional haplotype of CYP4F2 with increased transcriptional activity was associated with elevated urinary 20-HETE and hypertension in a Chinese population.6.Liu H. Zhao Y. Nie D. et al.Association of a functional cytochrome P450 4F2 haplotype with urinary 20-HETE and hypertension.J Am Soc Nephrol. 2008; 19: 714-721Crossref PubMed Scopus (55) Google Scholar A similar result was reported that the variant V433M in CYP4F2 was associated with the increase of 20-HETE excretion and systolic blood pressure (SBP) in a white cohort.7.Ward N.C. Tsai I.J. Barden A. et al.A single nucleotide polymorphism in the CYP4F2 but not CYP4A11 gene is associated with increased 20-HETE excretion and blood pressure.Hypertension. 2008; 51: 1393-1398Crossref PubMed Scopus (128) Google Scholar However, a different result showed that the variant F434S in CYP4A11, another human 20-HETE synthase gene, with reduced catalytic activity was associated with hypertension in white populations.8.Gainer J.V. Bellamine A. Dawson E.P. et al.Functional variant of CYP4A11 20-hydroxyeicosatetraenoic acid synthase is associated with essential hypertension.Circulation. 2005; 111: 63-69Crossref PubMed Scopus (172) Google Scholar Therefore, CYP4F2 transgenic approach would be a pivotal and irreplaceable strategy to clarify the correlation between 20-HETE and blood pressure. Heterologous promoters have been utilized to target transgene expression to expected tissues at controllable level in transgenic models. Native CYP4F2 is expressed at a high level in the proximal tubules of the human kidney where it accounts for the majority of renal 20-HETE production.1.Lasker J.M. Chen W.B. Wolf I. et al.Formation of 20-hydroxyeicosatetraenoic acid, a vasoactive and natriuretic eicosanoid, in human kidney. Role of CYP4F2 and CYP4A11.J. Biol Chem. 2000; 275: 4118-4126Crossref PubMed Scopus (267) Google Scholar Mouse kidney androgen-regulated protein (KAP) is one of the most abundant proteins expressed in the proximal tubules in response to different hormones such as androgen and estrogen.9.Meseguer A. Catterall J.F. Mouse kidney androgen-regulated protein messenger ribonucleic acid is expressed in the proximal convoluted tubules.Mol Endocrinol. 1987; 1: 535-541Crossref PubMed Scopus (48) Google Scholar Experimental evidence from many previous transgenic models has shown that a 1542-bp fragment of the KAP promoter could successfully regulate transgene expression in the kidney.10.Ding Y. Davisson R.L. Hardy D.O. et al.The kidney androgen-regulated protein promoter confers renal proximal tubule cell-specific and highly androgen-responsive expression on the human angiotensinogen gene in transgenic mice.J Biol Chem. 1997; 272: 28142-28148Crossref PubMed Scopus (112) Google Scholar, 11.Ding Y. Sigmund C.D. Androgen-dependent regulation of human angiotensinogen expression in KAP-hAGT transgenic mice.Am J Physiol Renal Physiol. 2001; 280: F54-F60Crossref PubMed Google Scholar, 12.Lavoie J.L. Lake-Bruse K.D. Sigmund C.D. Increased blood pressure in transgenic mice expressing both human renin and angiotensinogen in the renal proximal tubule.Am J Physiol Renal Physiol. 2004; 286: F965-F971Crossref PubMed Scopus (96) Google Scholar, 13.Malstrom S.E. Tornavaca O. Meseguer A. et al.The characterization and hormonal regulation of kidney androgen-regulated protein (Kap)-luciferase transgenic mice.Toxicol Sci. 2004; 79: 266-277Crossref PubMed Scopus (17) Google Scholar, 14.Sachetelli S. Liu Q. Zhang S.L. et al.RAS blockade decreases blood pressure and proteinuria in transgenic mice overexpressing rat angiotensinogen gene in the kidney.Kidney Int. 2006; 69: 1016-1023Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar Moreover, Soler's recent study has documented that a 224-bp truncated fragment of the KAP promoter was sufficient to drive androgen-dependent transactivation in the kidney.15.Soler M. Tornavaca O. Solé E. et al.Hormone-specific regulation of the kidney androgen-regulated gene promoter in cultured mouse renal proximal-tubule cells.Biochem J. 2002; 366: 757-766Crossref PubMed Google Scholar Herein, we chose the truncated KAP promoter to highlight kidney-dominant and inducible expression of CYP4F2 in transgenic mice. This study aims to develop a transgenic mouse model expressing CYP4F2 under the control of the heterologous KAP promoter, and to shed light on the impact of 20-HETE on blood pressure through CYP4F2 gain-in-function. To identify the KAP promoter activity, we amplified the 224-bp fragment from 5′-flanking of the KAP gene to construct pKAP-LUC and pKAP-CYP4F2/his expression vectors. The expression of luciferase and his-tagged CYP4F2 was detected after transient transfection into HEK293 cells. As illustrated in Figure 1a, the luciferase activity of cells transfected with the pKAP-LUC, after being normalized to Renilla luciferase activity, was threefold over those with the pGL3-enhancer control. Western blot analysis for CYP4F2 expression in cells transfected with the pKAP-CYP4F2/his (Figure 1b) showed 2.6-fold of cross-reacting signal over the pGL3-enhancer control using anti-CYP4F2 antibody, wherein endogenous CYP4F2 was included. The recombinant CYP4F2 expression was further verified by anti-his antibody. These results confirmed the 224-bp KAP promoter activity in driving the expression of the heterologous CYP4F2 gene in HEK293 cells. The linearized pKAP-CYP4F2/his (Figure 2a) was microinjected into FVB/N mouse to generate transgenic mice. We routinely screened the mice by PCR (data not shown) for the integration of CYP4F2 using specific primers, and further evaluated the presence of the transgene by Southern blot analysis (Figure 2b). Three transgenic founders, F0-6 (female), F0-16 (female), and F0-56 (male), were successfully bred to establish independent transgenic mouse lines. There was no difference in the number of offsprings between male and female, indicating the CYP4F2 gene may insert into the autosomes of the three founders. Line F0-16 seemed to be less reproducible and thereby the number of positive transgenic pups per brood was fewer than the others. CYP4F2 was expressed in all kidney samples of the three transgenic lines, among which line F0-16 exhibited the highest expression level (Figure 3a). Detailed expression profile of female and male transgenic offspring in line F0-16 was illustrated in Figure 3b and c, respectively, demonstrating a wide expression spectrum of CYP4F2. Densitometry scanning (Figure 3d) revealed that the highest expression level was in the kidney of male mice, about 2.5-fold over female littermates. In addition, the extra-renal expression of CYP4F2 was prominent in reproductive organs. These results indicate that human CYP4F2 was successfully expressed in the kidney of transgenic mice, and the expression was influenced by sexual hormones, especially by androgen. Immunohistochemistry was performed in the kidney, liver, brain, and uterus from line F0-16 using anti-his antibody. The immunostaining signal was abundant in the kidney and uterus, moderate in the liver, and not in the brain (Figure 4), which was consistent with the results of Western blot analysis. In the kidney, positive staining was predominantly localized in the proximal tubules, indicating a high level of CYP4F2 expression; and there was no immunostaining signal in the glomerulus. The liver sections exhibited positive immunostaining in some hepatocytes of the liver lobule. An intense staining could be observed throughout the uterus sections apart from the endomembrane. To address whether the overexpression of renal CYP4F2 was in correspondence to increased 20-HETE synthesis, we generated an AA hydroxylation reaction system in vitro. Renal microsomes isolated from transgenic line F0-16 and wild-type mice were incubated with AA. As illustrated in Figure 5a, the 20-HETE production was significantly higher in the transgenic group than in the wild-type group (660 (408–1073) versus 298 (224–398) pmol/min/mg microsome, P=0.003). Moreover, Western blot analysis for CYP4F2 in the renal microsomes demonstrated that the cross-reacting signal of transgenic mice was 3.5-fold over wild-type controls (Figure 5b). From these results, we conclude that the overexpressed CYP4F2 in the renal microsomes contributed to increased 20-HETE production in transgenic mice. The urinary 20-HETE level of transgenic mice was determined to evaluate the activity of CYP4F2 in vivo. 20-HETE ELISA assay was performed for the measurement according to Grates's report.16.Grates H.E. McGowen R.M. Gupta S.V. et al.Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay.J Biosci. 2003; 28: 109-113Crossref PubMed Scopus (16) Google Scholar As shown in Table 1, both female and male transgenic mice from all the independent lines had increased urinary 20-HETE compared with wild-type controls (P<0.05). The urinary 20-HETE of male mice from line F0-16 was the highest of all. Male transgenic mice had higher urinary 20-HETE than their female littermates (P=0.046), which was in accordance with higher renal CYP4F2 expression in male than in female mice. These results demonstrate that the overexpressed CYP4F2 in transgenic mice functioned and lead to an augment in urinary 20-HETE excretion.Table 1Urinary 20-HETE of CYP4F2 transgenic miceGroupn20-HETE (ng/ml)P-valueWT Female68.99 (3.17–25.47) Male712.45 (7.88–19.66)Line F0-16 Female640.23 (22.43–72.16)0.0001 Male673.28 (42.23–127.18)0.000005Line F0-56 Female833.27 (20.67–53.53)0.0003 Male735.55 (23.95–52.77)0.003Line F0-6 Female723.91 (14.24–40.13)0.007 Male1041.36 (25.40–67.31)0.0003WT, wild-type.As urinary 20-HETE was not normally distributed, it underwent logarithmic transformation for analysis of variance and was shown as geometric mean (95% CI). Open table in a new tab WT, wild-type. As urinary 20-HETE was not normally distributed, it underwent logarithmic transformation for analysis of variance and was shown as geometric mean (95% CI). To determine the effect of 20-HETE overproduction on blood pressure, we measured the mouse blood pressure by external tail pulse detection. As depicted in Table 2, the SBP of transgenic mice was elevated with variable magnitude compared with wild-type controls. The most conspicuous increment of SBP was in line F0-16. Figure 6a better shows the comparison. Male transgenic mice had significantly higher level of SBP than female transgenic littermates (P=0.001). The changes in SBP basically paralleled the changes in urinary 20-HETE shown in Table 1, and their positive correlation was shown in Figure 6b with statistical significance (R=0.872, P=0.005), indicating that the 20-HETE overproduction contributed to elevated blood pressure in transgenic mice. To record the development of hypertension in transgenic mice, we performed time course measurement and found that the blood pressure was elevated since early lives and was maintained at a high level throughout late lives (Figure 7). In addition, the hypertensive phenotype of transgenic mice was further verified by intra-arterial blood pressure measurement (Supplementary Information).Table 2SBP of CYP4F2 transgenic miceGroupnSBP (mm Hg)P-valueWT Female12109.47±7.79 Male10117.06±8.11Line F0-16 Female8123.30±4.330.00008 Male7135.49±3.020.000002Line F0-56 Female11123.18±6.950.00002 Male11129.36±5.590.0002Line F0-6 Female6112.83±7.08NS Male9122.56±10.93NSWT, wild-type; NS, not significant.Data are presented as mean±s.d. Open table in a new tab Figure 7Time course blood pressure measurement. The blood pressure of transgenic (TG) mice in line F0-16 (n=12) and wild-type (WT) controls (n=9) aged 8 to 40 weeks was measured at four-week intervals to record the development of hypertension. The SBP of TG mice was elevated in early life and maintained at higher levels than WT controls throughout late life.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Download .jpg (.03 MB) Help with files Supplementary Information WT, wild-type; NS, not significant. Data are presented as mean±s.d. Finally, we performed blood biochemical assays including serum creatinine, blood urea nitrogen (BUN), and plasma aldosterone determination to exclude some other factors associated with the development of hypertension. As shown in Table 3, all these parameters were at equal levels between transgenic mice and wild-type controls. Therefore, the development of hypertension in our model was not related to kidney failure or hyperaldosteronism, which further confirmed the causative role of 20-HETE in the development of hypertension.Table 3Blood biochemical assays of CYP4F2 transgenic miceParameterWT (n=6)TG (n=5)P-valueCreatinine (mg per 100 ml)0.31±0.160.39±0.130.372BUN (mg per 100 ml)28.54±3.9427.78±0.990.664Aldosterone (pg per 100 ml)335.60±92.41321.47±85.120.800TG, transgenic; WT, wild-type.Data are presented as mean±s.d. Open table in a new tab TG, transgenic; WT, wild-type. Data are presented as mean±s.d. This study offers a novel transgenic mouse model overexpressing human CYP4F2 under the control of the heterologous KAP promoter. CYP4F2 was proved to be overexpressed, leading to an augment of 20-HETE production, which, in consequence, contributed to elevated blood pressure in CYP4F2 transgenic mice. Native CYP4F2 is predominantly expressed in the kidney and plays a pivotal role in the regulation of blood pressure through the vasoactive and natriuretic effects of 20-HETE metabolite.2.McGiff J.C. Quilley J. 20-HETE and the kidney: resolution of old problems and new beginnings.Am J Physiol. 1999; 277: R607-R623Crossref PubMed Google Scholar, 3.Miyata N. Roman R.J. Role of 20-hydroxyeicosatetraenoic acid (20-HETE) in vascular system.J Smooth Muscle Res. 2005; 41: 175-193Crossref PubMed Scopus (194) Google Scholar, 4.Roman R.J. P-450 metabolites of arachidonic acid in the control of cardiovascular function.Physiol Rev. 2002; 82: 131-185Crossref PubMed Scopus (1169) Google Scholar, 5.Sarkis A. Roman R.J. Role of cytochrome P450 metabolites of arachidonic acid in hypertension.Curr Drug Metab. 2004; 5: 245-256Crossref PubMed Scopus (73) Google Scholar Based on the anatomical and functional characters of CYP4F2 in the kidney, the heterologous KAP promoter was used to control CYP4F2 expression in a kidney-dominant pattern in transgenic mice. CYP4F2 was successfully expressed in the kidney of transgenic mice, and its renal localization was quite similar to the spatial disposition of native CYP4F2 in the human kidney. To assess the activity of the overexpressed CYP4F2, we performed the hydroxylation assay in isolated renal microsomes. We found 20-HETE production was significantly higher in samples isolated from transgenic mice than in those from control mice, and the elevated 20-HETE production was in respondence to the increased CYP4F2 expression level in isolated renal microsomes from transgenic mice, indicating the overexpressed CYP4F2 resulted in a high level of AA ω-hydroxylation into 20-HETE. The elevated urinary 20-HETE excretion could further substantiate this notion in vivo. In addition, the CYP4F2 expressed in other extra-renal tissues may also contribute to some extent to the increased 20-HETE level in the systemic circulation, which in return exacerbated urinary 20-HETE excretion. The quantification of 20-HETE in this study was performed using Grates's 20-HETE ELISA method,16.Grates H.E. McGowen R.M. Gupta S.V. et al.Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay.J Biosci. 2003; 28: 109-113Crossref PubMed Scopus (16) Google Scholar by which the 20-HETE concentration was comparatively higher than that by GC-MS techniques17.Holla V.R. Adas F. Imig J.D. et al.Alterations in the regulation of androgen-sensitive cyp 4a monooxygenase cause hypertension.Proc Natl Acad Sci USA. 2001; 9: 5211-5216Crossref Scopus (218) Google Scholar and was somewhat lower than that by fluorescent HPLC analysis.18.Maier K.G. Henderson L. Narayanan J. et al.Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachidonic acid.Am J Physiol Heart Circ Physiol. 2000; 279: H863-H871Crossref PubMed Google Scholar However, the comparison between transgenic and their wild-type littermates under the same background was valid to reflect the effects of the overexpressed CYP4F2. The aforementioned data suggest that the transgenic model with an overexpression of CYP4F2 increased in 20-HETE production, and could be used for clarifying the effect of CYP4F2 on blood pressure through the 20-HETE pathway. 20-HETE has both prohypertensive and antihypertensive properties.2.McGiff J.C. Quilley J. 20-HETE and the kidney: resolution of old problems and new beginnings.Am J Physiol. 1999; 277: R607-R623Crossref PubMed Google Scholar, 3.Miyata N. Roman R.J. Role of 20-hydroxyeicosatetraenoic acid (20-HETE) in vascular system.J Smooth Muscle Res. 2005; 41: 175-193Crossref PubMed Scopus (194) Google Scholar, 4.Roman R.J. P-450 metabolites of arachidonic acid in the control of cardiovascular function.Physiol Rev. 2002; 82: 131-185Crossref PubMed Scopus (1169) Google Scholar, 5.Sarkis A. Roman R.J. Role of cytochrome P450 metabolites of arachidonic acid in hypertension.Curr Drug Metab. 2004; 5: 245-256Crossref PubMed Scopus (73) Google Scholar 20-HETE promotes hypertension by vasoconstriction in many microcirculatory districts that regulates the vascular tone and homeostasis. On the contrary, 20-HETE attenuates hypertension by the inhibition of ion reabsorption that regulates the salt balance. In our study, two lines of transgenic mice (F0-16 and F0-56) had significantly higher SBP than the control. Transgenic line F0-16 with the highest renal CYP4F2 expression and urinary 20-HETE had the highest SBP level, which could likely account for the lowest reproductivity in this line; whereas line F0-6 with relatively low level of CYP4F2 and urinary 20-HETE failed to change blood pressure. The average SBP positively correlated with urinary 20-HETE level with statistical significance. Thus, our results supported that a prohypertensive effect of 20-HETE was in dominance. After excluding some other factors associated with the development of hypertension, such as kidney failure and hyperaldosteronism, we deduce 20-HETE derived from the overexpressed CYP4F2 in the tubular system of nephron may diffuse into the local and systemic circulation where the vasoactive molecule elicited powerful vasoconstriction action in the transgenic mice. In addition, 20-HETE synthesized in extra-renal organs may also increase the peripheral vascular tone. The prohypertensive effect of 20-HETE was evident in the early lives of transgenic mice and lasted throughout their late lives. As to the antihypertensive action of 20-HETE by inhibiting salt reabsorption, we presumed it was not overt enough to override the prohypertensive effect. Previous investigations indicated that 20-HETE plays an important role in the renal adaptation to elevated Na+ intake: Laffer et al.19.Laffer C.L. Laniado-Schwartzman M. Wang M.H. et al.Differential regulation of natriuresis by 20-hydroxyeicosatetraenoic Acid in human salt-sensitive versus salt-resistant hypertension.Circulation. 2003; 107: 574-578Crossref PubMed Scopus (90) Google Scholar demonstrated that urinary 20-HETE excretion was 66% higher during salt loading than during salt depletion in humans; another study also showed that urinary 20-HETE excretion increased by 20% when rats were fed a high salt diet.20.Hoagland K.M. Flasch A.K. Roman R.J. Inhibitors of 20-HETE formation promote salt-sensitive hypertension in rats.Hypertension. 2003; 42: 669-673Crossref PubMed Scopus (79) Google Scholar In this regard, we hypothesized that our transgenic mice fed with normal salt diet did not elicit strong ion reabsorption inhibition. The prohypertensive effect of 20-HETE in our transgenic model through CYP4F2 gain-in-function was in agreement with our recent report that a functional haplotype of CYP4F2 with increased transcriptional activity was associated with elevated urinary 20-HETE and enhanced susceptibility of hypertension,6.Liu H. Zhao Y. Nie D. et al.Association of a functional cytochrome P450 4F2 haplotype with urinary 20-HETE and hypertension.J Am Soc Nephrol. 2008; 19: 714-721Crossref PubMed Scopus (55) Google Scholar and was also in agreement with Ward et al.'s report7.Ward N.C. Tsai I.J. Barden A. et al.A single nucleotide polymorphism in the CYP4F2 but not CYP4A11 gene is associated with increased 20-HETE excretion and blood pressure.Hypertension. 2008; 51: 1393-1398Crossref PubMed Scopus (128) Google Scholar that the variant V433M in CYP4F2 was associated with the increase of 20-HETE excretion and SBP. Many previous animal models also supported that increased renal CYP activity and 20-HETE promoted hypertension: the delivery of CYP4A1 cDNA into Sprague–Dawley rat increased blood pressure, and the transfer of antisense CYP4A1 cDNA into spontaneously hypertensive rat attenuated the development of hypertension;21.Zhang F. Chen C.L. Qian J.Q. et al.Long-term modifications of blood pressure in normotensive and spontaneously hypertensive rats by gene delivery of rAAV-mediated cytochrome P450 arachidonic acid hydroxylase.Cell Res. 2005; 15: 717-724Crossref PubMed Scopus (23) Google Scholar other researches on spontaneously hypertensive rat reported that the CYP4A2 gene was overexpressed in the kidney,22.Iwai N. Inagami T. Isolation of preferentially expressed genes in the kidneys hypertensive rats.Hypertension. 1991; 17: 161-169Crossref PubMed Scopus (159) Google Scholar with increased renal 20-HETE, and the use of 20-HETE synthesis inhibitor attenuated hypertension.23.Su P. Kaushal K.M. Kroetz D.L. Inhibition of renal arachidonic acid omega-hydroxylase activity with ABT reduces blood pressure in the SHR.Am J Physiol. 1998; 275: R426-R438PubMed Google Scholar In addition, the CYP4F2 expression in our transgenic mice was found in a wide spectrum and was prominent in reproductive organs other than in the kidney. The extra-renal transgene expression was also found in other transgenic models controlled by the 1542-bp KAP promoter: Ding's investigation of KAP-hATG transgenic mice revealed that human angiotensinogin was restricted to the kidney and epididymis of a male line10.Ding Y. Davisson R.L. Hardy D.O. et al.The kidney androgen-regulated protein promoter confers renal proximal tubule cell-specific and highly androgen-responsive expression on the human angiotensinogen gene in transgenic mice.J Biol Chem. 1997; 272: 28142-28148Crossref PubMed Scopus (112) Google Scholar and was induced by testosterone treatment in female transgenic mice;11.Ding Y. Sigmund C.D. Androgen-dependent regulation of human angiotensinogen expression in KAP-hAGT transgenic mice.Am J Physiol Renal Physiol. 2001; 280: F54-F60Crossref PubMed Google Scholar human renin was expressed in the kidney of all KAP-hREN transgenic lines and was variable in other tissues;12.Lavoie J.L. Lake-Bruse K.D. Sigmund C.D. Increased blood pressure in transgenic mice expressing both human renin and angiotensinogen in the renal proximal tubule.Am J Physiol Renal Physiol. 2004; 286: F965-F971Crossref PubMed Scopus (96) Google Scholar the reporter luciferase gene displayed high expression levels in the kidney and reproductive organs of KAP-luciferase transgenic mice.13.Malstrom S.E. Tornavaca O. Meseguer A. et al.The characterization and hormonal regulation of kidney androgen-regulated protein (Kap)-luciferase transgenic mice.Toxicol Sci. 2004; 79: 266-277Crossref PubMed Scopus (17) Google Scholar The CYP4F2 expression was higher and wider in our model than in those previous models. This may be explained by the truncation of the 224-bp promoter, or by the potential role of the introduced SV40 enhancer in the pKAP-CYP4F2/his construct as enhancer element was suggested to be required for the KAP promoter activity.24.Bianco R.A. Keen H.L. Lavoie J.L. et al.Untraditional methods for targeting the kidney in transgenic mice.Am J Physiol Renal Physiol. 2003; 285: F1027-F1033Crossref PubMed Scopus (10) Google Scholar As to the prominent expression of CYP4F2 in reproductive organs of the transgenic mice, it may be explained that the truncated KAP promoter was capable of driving sex hormone responsive expression as native KAP is expressed in response to different hormones, such as androgen and estrogen.25.Meseguer A. Catterall J.F. Cell-specific expression of kidney androgen-regulated protein messenger RNA is under multihormonal control.Mol Endocrinol. 1990; 4: 1240-1248Crossref PubMed Scopus (39) Google Scholar Furthermore, we also found sex-dimorphous phenomena in CYP4F2 expression, renal 20-HETE formation and SBP in our transgenic model. The transgene expression in the kidney was higher in male than in female mice; urinary 20-HETE was higher in male transgenic mice than in female littermates; and coincidently the average SBP was significantly higher in male transgenic mice than in female littermates. As to the nontransgenic mice, 20-HETE and SBP had male mice dominance as well. The disparities between gender in our study may be explained from two aspects: one was that the 224-bp KAP promoter regulated CYP4F2 expression in an androgen-responsive way; the other was that androgen-mediated 20-HETE synthesis directly. Several animal models have suggested a link between 20-HETE production and androgen: Holla et al.17.Holla V.R. Adas F. Imig J.D. et al.Alterations in the regulation of androgen-sensitive cyp 4a monooxygenase cause hypertension.Proc Natl Acad Sci USA. 2001; 9: 5211-5216Crossref Scopus (218) Google Scholar found that androgen can induce the expression of mice cyp4a12, 20-HETE formation and hypertension; Sprague–Dawley rats showed an increase in the renal 20-HETE and SBP level after androgen induction.26.Nakagawa K. Marji J.S. Schwartzman M.L. et al.The androgen-mediated induction of the kidney arachidonate hydroxylases is associated with the development of hypertension.Am J Physiol Regul Integr Comp Physiol. 2003; 284: R1055-R1062Crossref PubMed Scopus (86) Google Scholar Our previous population study including 132 participants had shown that the urinary 20-HETE level in men was slightly higher than in women (P=0.041).6.Liu H. Zhao Y. Nie D. et al.Association of a functional cytochrome P450 4F2 haplotype with urinary 20-HETE and hypertension.J Am Soc Nephrol. 2008; 19: 714-721Crossref PubMed Scopus (55) Google Scholar Another research involving 66 subjects documented that men had significantly higher levels of urinary 20-HETE than women (P=0.007).27.Ward N.C. Rivera J. Hodgson J. et al.Urinary 20-hydroxyeicosatetraenoic acid is associated with endothelial dysfunction in humans.Circulation. 2004; 110: 438-443Crossref PubMed Scopus (98) Google Scholar In addition, a recent study also reported men had significantly higher levels of 20-HETE excretion when compared with women.7.Ward N.C. Tsai I.J. Barden A. et al.A single nucleotide polymorphism in the CYP4F2 but not CYP4A11 gene is associated with increased 20-HETE excretion and blood pressure.Hypertension. 2008; 51: 1393-1398Crossref PubMed Scopus (128) Google Scholar Taken together, our findings suggested that androgen partially contributed to the overexpressed CYP4F2, increased 20-HETE production and elevated blood pressure. In summary, we established for the first time a CYP4F2 transgenic mouse model. The overexpression of CYP4F2 successfully participated in the overproduction of 20-HETE, and consequently resulted in elevated arterial blood pressure. The novel CYP4F2 transgenic model provides a valid tool to further elucidate the mechanism of CYP4F2–20-HETE pathway on blood pressure. The promoter fragment from -224 to +1 of the KAP gene was obtained by PCR using mouse genomic DNA as template and primers: 5′-GCGGTACCGGGTGGTGCACCTAGTCAG-3′ and 5′-CCAGATCTCCAAAAAGAGTCCTCCTGG-3′. The amplified product was cloned into the KpnI /HindIII sites of the pGL3-enhancer vector (Promega, Madison, WI, USA) to generate pKAP-LUC plasmid. The CYP4F2 cDNA was amplified by PCR with primers 5′-TGCCCTGCAGGATGTCCCAGCTGA-3′ and 5′-CTCTCTAGATCAATGATGATGATGATGATGGCTCAGGGGCTCCACCCGCAG-3′ (an introduced 6 × his tag was underlined) after reverse transcription of human liver RNA. The amplified CYP4F2 cDNA was cloned into the PstI / XbaI sites of the pKAP-LUC to generate pKAP-CYP4F2/his construct. All constructs were confirmed with sequencing. Kidney-derived human embryo kidney 293 (HEK293) cells were cultured and co-transfected with the pKAP-LUC construct and the pRL-TK plasmid (Promega), encoding Renilla luciferase as internal control for luciferase assay, or transfected with the pKAP-CYP4F2/his construct for Western blot analysis, using lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Cells were harvested 24 h after transfection. Luciferase assay was performed using Dual Luciferase Reporter assay system (Promega) and a Lumat LB 9507 luminometer (Bethold Technologies, Bad Wildbad, Germany). The pKAP-CYP4F2/his plasmid was digested by KpnI and BamHI to a linear DNA fragment containing the 224-bp KAP promoter, CYP4F2 cDNA and SV40 enhancer, and microinjected into FVB/N mouse one-cell fertilized embryos to generate CYP4F2 transgenic founders (Figure 3a). Mice were fed with standard mouse chow and water ad libitum, and bred in a 12:12 h light–dark cycle system. Transgenic mice of 14- to 20-week old were used for data collection unless noted elsewhere, with age- and sex-matched wild-type mice as controls. All experiments conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Transgenic mice were identified by PCR and Southern blot. PCR was performed using DNA isolated from tail biopsies with primers 5′-TGGACCTACGCCTTCTATGA-3′ and 5′-CCACTTGTCACCAGCACTCA-3′ specific to human CYP4F2 cDNA. For Southern blot, the CYP4F2 cDNA sequence was isolated from the pKAP-CYP4F2/his by digestion with PstI and XbaI, labeled by [a-32P]dCTP (Furui Biotechnology, Beijing, China) with random primer DNA labeling kit (TaKaRa Biotech, Japan), and then hybridized with genomic DNA digested with HindIII. Total protein was extracted from transfected cells or tissues of transgenic mice and the concentration was determined with the Bradford method. Denatured protein was separated by electrophoresis and transferred onto poly (vinylidene fluoride) membranes at 4°C. Membranes were subsequently incubated with anti-CYP4F2 antibody (1:1000) (Fitzgerald, Concord, MA, USA), anti-his antibody (1:1000) (Sigma-Aldrich, St Louis, MO, USA), or anti-GAPDH antibody (1:10,000) (KangChen Bio-Tech, Shanghai, China) as primary antibody, followed by alkaline phosphatase-conjugated IgG (1:2000) as the secondary antibody. The final detection reaction was performed with β-naphthyl acid phosphate and Fast Blue B salt (Sigma-Aldrich). Mice were killed and immediately perfused transcardiacally with ice-cold PBS, followed by 4% paraformaldehyde in PBS. Organs were isolated, fixed at 4°C for additional 4 h, and then immersed in 30% sucrose solution at 4°C overnight. The samples were embedded in low temperature paraffin wax, and 4-μm thick sections were then prepared and incubated with anti-his antibody (1:300) at room temperature for 6 h. With Streptavitin Peroxidase-Conjugated (SP) method, Diaminobenzidin (Maxmim Bio-Tech, Fuzhou, China) was utilized as the chromagen to localize peroxidase activity. Photomicrographs were taken by an OLYMPS IX51 inverted microscope with the OLYMPUSMicro software. Renal microsomes were prepared according to the method described before1.Lasker J.M. Chen W.B. Wolf I. et al.Formation of 20-hydroxyeicosatetraenoic acid, a vasoactive and natriuretic eicosanoid, in human kidney. Role of CYP4F2 and CYP4A11.J. Biol Chem. 2000; 275: 4118-4126Crossref PubMed Scopus (267) Google Scholar for Western blot analysis and AA hydroxylation assay. The conversion of AA to 20-HETE was assessed in a reaction mixture of 300 μl volume containing 100 mM potassium phosphate buffer (pH 7.4), 3.3 mM MgCl2, 80 μM AA (Cayman Chemical, Ann Arbor, MI, USA), 1 mM NADPH (Roche Applied Science, Basel, Switzerland) and 0.6 μg/μl mouse renal microsomes. After violent vortex and 5 min preincubation at 37°C, NADPH was added to start the reaction at 37°C for 30 min, then the 20-HETE production was measured. 20-HETE ELISA kits (Detroit R&D, Detroit, MI, USA) were used in the AA hydroxylation assay of renal microsomes and urinary 20-HETE measurement following manufacturer's instructions. Briefly, samples were loaded to the 96-well plate that was coated with anti-20-HETE antibody. The plate was read at 450 nm OD using an automatic ELISA plate reader (Bio-Rad Model 680, Hercules, CA, USA), and the 20-HETE concentrations were calculated from the standard curve. The blood pressure of conscious mice was measured by tail-cuff method using IITC Life Science Model 1631 tail pulse detection system (IITC Life Science Model 1631, Woodland Hills, CA, USA) according to the manufacturer's instructions. Mice were trained for 5 days and acclimated mice were warmed at 33°C in the heating container for 5 min before measurement. One measurement session involved 10 repetitions and at least three sessions were performed on each mouse to calculate the average. Blood samples were collected for biochemical assays including serum creatinine, BUN, and plasma aldosterone determination. Serum creatinine and BUN measurement was performed on the Vitros 950 analyzer (Ortho-Clinical Diagnostics, Raritan, NJ, USA) with the VITROS Chemistry Products CREA and BUN Slides according to the manufacturer's instructions. Plasma aldosterone was determined by radioimmunoassay (Active Aldosterone Ria Kit, DSL 8600, Diagnostic System Laboratories, Webster, TX, USA) using Gama Immunoassay Counter (USTC Chuangxin Co., Ltd. Zonkia Branch, Hefei, China). Statistical analysis was run with SPSS version 10.0 for Windows (SPSS, Chicago, IL, USA). 20-HETE concentration was not normally distributed and was presented by geometric mean (95% CI). Mouse SBP, serum creatinine, BUN, and plasma aldosterone were presented by mean±s.d. Comparison between logarithmic urinary 20-HETE and SBP was performed by one-way ANOVA, and their correlation was identified by Pearson's method. Serum creatinine, BUN, and plasma aldosterone were compared by independent-sample t-test. Statistical significance was defined at P<0.05. This study was supported by grants from the National Natural Science Foundation of China (30571027) and the Natural Science Foundation of Liaoning Province (2007225004-4). Supplementary information accompanies the paper on the Kidney International website (http://www.nature.com/ki).
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