Research On CYP4F2/20-HETE Pathway In CYP4F2 Transgenic Mouse Model

IntroductionCytochrome P450 4F2 (CYP4F2) encodes a major 20-hydroxyeicosatetraenoic acid (20-HETE) synthase in human kidney that converts arachidonic acid (AA) into 20-HETE throughω-hydroxylation.20-HETE participates in the development of hypertension by regulating vascular and renal tubular functions. On the one hand, 20-HETE promotes hypertension by vasoconstriction or by sensitizing the vascular smooth muscle cells to other vasoconstrictors. On the other hand,20-HETE attenuates hypertension by inhibiting sodium reabsorption in the renal proximal tubles and ascending thich limb of Henle's Loop. Our group have rescently found that a functional haplotype of CYP4F2 with increased transcriptional activity was associated with elevated urinary 20-HETE and hypertension in Han Chinese residing in a hypertension-prevalent region of Liaoning Province. 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 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. Therefore, CYP4F2 transgenic approach would be a pivotal and irreplaceable strategy to clarify the correlation between 20-HETE and blood pressure at the level of whole organism.Kidney androgen-regulated protein (KAP) is one of the most abundant proteins expressed in mouse proximal tubule that resembles the expression locus of CYP4F2 in human kidney. KAP promoter activity is regulated by different hormones such as androgen and estrogen, among which androgen shows the strongest responsibility. Previous KAP-hAGT and KAP-LUC transgenic models have shown that KAP promoter could successfully regulate androgen-inducible transgene expression in the kidney. Herein, we constructed CYP4F2 transgenic mouse model utilizing KAP promoter to to highlight CYP4F2 expression in the kidney where the dual role of 20-HETE fulfils, and to better understand the mechanism of CYP4F2/20-HETE in blood pressure regulation by androgen induction.The present study developed a transgenic mouse model expressing CYP4F2 under the control of the heterologous KAP promoter to shed light on the impact of CYP4F2/20-HETE pathway on blood pressure at the level of whole organism. With androgen as an inducer of CYP4F2 expression, further studies based on this model would help to gain insight into the detailed mechanism of CYP4F2/20-HETE in blood pressure regulation.Materials and MethodsMaterials1.FVB/N mice2. Human cell line HEK293 and HUVEC3. Reagents for gene cloning and luciferase assay4. Reagents for Southern blot, Western blot and immunohistochemistry5. Reagents for 20-HETE ELISA and aldosterone RIA determination6. Reagents for Real-time PCR assay7. ROS-specific fluorescent dye DHE and NO-specific fluorescent dye DAF-FM8. Reagents for MDA and SOD activity assay9. Microarrays of oxidative stress gene mRNA expressionMethods 1. Kidney-derived HEK293 cells were transfected with constructed pKAP-LUC, pKAP-CYP4F2/his expression plasmid. Luciferase activity and CYP4F2 expression were determined to identify the promoter activity of KAP promoter in cultured cells.2. The linearized pKAP-CYP4F2/his was microinjected into FVB/N mouse one-cell fertilized embryoes to generate transgenic founders. Tail DNA was isolated for Southern blot and PCR identification of positive mice with probes and primers designed specific to CYP4F2.3. Total protein from various tissus including kidney was extracted and determined concentration. The expression profile of CYP4F2 was determined by Western blot using anti-his antiby. The 4% paraformaldehyde fixed kidney samples were embedded by paraffin wax and sectioned for immunohistochemistry to localize the CYP4F2 expression. Renal microsomes were also extracted to test CYP4F2 expression by Western blot.4. The in vitro AA hydroxylation assay was assessed by incubating the AA substrate, NADPH coenzyme, etc. with the renal microsomes.20-HETE production was quantified by specific 20-HETE enzyme-linked immunosorbent assay (ELISA) kit. Mice urine was collected to measure the level of 20-HETE by ELISA kit.5. Blood pressure of transgenic mice was measured by noninvasive tail-cuff method as well as carotid catheter. Mice aging from 8 to 40 weeks were measured to record the time course of hypertension development. In addition, blood sample was collected from orbital sinus for plasma and serum separation. Plasma aldosterone was determined by radioimmunoassay (RIA), and serum creatinine (CRE) and blood urea nitrogen (BUN) were measured using CREA and BUN Slides.6. Female mice were i.p. injected with 5a-dihydrotestosterone (DHT) for consecutive 14 days. After DHT induction, renal CYP4F2 expression was examined by Western blot.20-HETE level was measured by ELISA in the urine as well as in the microsomal AA hydroxylation reaction mixture. Mouse blood pressure was measured by tail-cuff method. 7. Male mice underwent castration to clear endogenous androgens before exogenous DHT administration. The level of renal CYP4F2 expression,20-HETE and SBP was also measured accordingly.8. Mice furthermore underwent coadministration of either DHT with HET0016, the specific 20-HETE inhibitor, or DHT with lecithin vehicle as control. HET0016 was applied to confirm the role of 20-HETE in the development of androgen-induced hypertension.9. Total RNA from the DHT-induced mouse kidney was extracted and reverse-transcripted into cDNA. DHT regulation on mouse endogenouse 20-HETE synthase, cyp4a12, cyp4a14, and cyp4a10, was studied by Real-time PCR.10. Human HEK293 cells were treated with DHT for 0.5 hour,24 hours and 48 hours, respectively. CYP4F2 exptression was examined by Western blot analysis using anti-CYP4F2 antibody.11. The level of oxidative stress in mice was evaluated by relevant markers:the level of reactive oxygen species (ROS) was in situ examined in the frozen sectioned thoracic aorta and kidney; plasma and renal homogenate malondialdehyde (MDA) was evaluated as a marker of lipid peroxidation; plasma and renal homogenate total superoxide dismutase (SOD) was measured as a major antioxidative parameter.12. Mice were treated with Tempol, an SOD mimetic to relieve oxidative stress, and the SBP was measured by tail-cuff method.13. Total renal RNA was extracted and reverse-transcripted into cDNA, and the microarray expression analysis including 84 oxidative stress genes was preformed using Real-time PCR.14. Nos2, one of the differentially expressed genes screened by microarray analysis, was chosen as candidate for detailed analysis. The renal expression of iNOS, nNOS, and eNOS was further confirmed by Western blot.15. HUVEC and HEK293 cells were cultured and stimulated by 20-HETE. The level of oxidative stress was studied by ROS-specific fluorescent dye DHE, and the level of NO was determined by the fluorescent dye DAF-FM.ResultsⅠ. Generation and identification of CYP4F2 transgenic mouse model1. Kidney-derived HEK293 cells transfected with pKAP-LUC showed increased luciferase activity by 3-fold over control, and cells with pKAP-CYP4F2/his plasmid expressed CYP4F2 of 2.6-fold higher than control.2. Three CYP4F2 transgenic founders, generated by microinjection, were identified by Southern blot and PCR Therefore, three transgenic lines were established after breeding, namely F0-6, F0-16, and F0-56.3. Renal CYP4F2 expression was the highest in line F0-16, and was also positive in the other two transgenic lines. The expression profile demonstrated that:CYP4F2 was expressed at the highest level in male kidney; in addition, the expression was also high in sexual-hormone responsive organs such as testis, epididymis, uterus, and ovary.4. In CYP4F2 transgenic mice, renal CYP4F2 mainly localized in the proximal tubules, and CYP4F2 expression and AA hydroxylation were increased in the renal microsomes compared with wild-type controls. Transgenic mice also had significantly higher level of urinary 20-HETE excretion than wild-type mice.5. The systolic blood pressure (SBP) in transgenic mice was obviously elevated than in wild-type control, and was in positive correlation with urinary 20-HETE excretion. Hypertension developed in early lives of transgenic mice. Plasma aldosterone, serum CRE and BUN were not different between transgenic mice and wild-type controls.Ⅱ. Androgen effect on CYP4F2 transgenic mouse model1. Renal CYP4F2 expression was further up-regulated after DHT induction in female transgenic mice. Consequently,20-HETE production was enhanced. The blood pressure in transgenic mice was furthermore elevated after induction.2. Male transgenic mice showed decreased renal CYP4F2 expression after castration, and exhibited dramaticly increased expression after exogenous DHT induction.20-HETE excretion and SBP accompanied the changes in androgen level, i.e., first declined, and then elevated.3. Coadministration of DHT and 20-HETE specific inhibitor HET0016 negated DHT induced 20-HETE excretion and hypertension. This further confirmed the contributive role of 20-HETE in the development of androgen-induced hypertension.4. All the three isoforms of mouse endogenouse 20-HETE synthases were greatly depressed and were less sensitive to DHT in the kidney of CYP4F2 transgenic mice compared to wild-type controls. The mRNA expression of cyp4a12 was up-regulated by androgen, and cyp4al4 was down-regulated by androgen. Nontheless, cyp4a10 responded to androgen with no statistical significance.5. Human HEK293 cells incubated with DHT for 0.5 hour,24 hours and 48 hours, showed increased CYP4F2 exptression of 1.4-fold,2.3-fold and 2.5-fold over control, respectively, indicating a possibility that CYP4F2 was androgen-dependent in human kidney.Ⅲ. Oxidative stress of CYP4F2 transgenic mouse model1. The level of ROS in the frozen sectioned thoracic aorta and kidney was obviously higher in CYP4F2 transgenic mice than in control. Meanwhile, plasma and renal homogenate MDA was elevated, whereas total SOD activity was decreased in transgenic mice.2. Transgenic mice administered with Tempol significantly lessened the degree of hypertensive phenotype to nearly normal level, indicating a key role of oxidative stress in hypertension pathogenesis.3. Microarray expression analysis of oxidative stress genes revealed 23 differentially expressed genes at the significance level of P＜0.05, and 12 genes at the significance level of P＜0.01. 4. After gene screening, iNOS, as well as nNOS and eNOS, were chosen as candidate for further analysis. Their expressions in mice kidney were confirmed by Western blot, displaying the increased iNOS and the decreased nNOS in CYP4F2 transgenic mice compared to wild-type controls.5. HEK293 cells stimulated with 20-HETE showed significantly higher level of ROS determined by DHE fluorescent dye. There is no obvious change of NO with 20-HETE as stimulus using DAF-FM fluorescent dye.Conclusions1. In present study, we successfully established a novel CYP4F2 transgenic mouse model. Transgenic mice with gain-in-function of CYP4F2/20-HETE metabolic pathway showed chronic hypertensive phenotype. Therefore, we provided evidence that the CYP4F2/20-HETE played a pro-hypertensive role at the level of whole organism.2. Androgen strongly induced the renal CYP4F2 expression and magnified the bioeffect of CYP4F2/20-HETE pathway, which thereby caused androgen-responsive hypertension. In addition, the androgen-inducibility of CYP4F2 in human kidney cells further indicated a possible mechanism of CYP4F2/20-HETE underlying gender-specific differences of human hypertension.3. CYP4F2 transgenic mice suffered severe oxidative stress, and anti-oxidative treatment ameliorated the hypertensive phenotype. In addition, the expression of oxidative stress genes extensively varied between transgenic and wild-type mice, suggesting oxidative stress was the key point in the pathogenesis of hypertension, and was involed in the pro-hypertensive mechanism of CYP4F2/20-HETE pathway.