Any patient with HT diagnosis (systolic and/or a diastolic blood pressure values ≥140 and ≥90mm Hg, respectively) referred to the Service of Nephrology from “Virgen del Puerto” Hospital (Plasencia, Spain) who was ≥18 years old was eligible for recruitment. A group of 151 HT patients (62.7% males; 68.0±13.6 years) were finally enrolled in the study.

Additionally, a group of 87 normotensive (NT; systolic and/or a diastolic blood pressure reading<140 and <90mm Hg, respectively) subjects (74.7% females; 28.1±11.3 years) ≥18 years old, mainly students and staff from the University of Extremadura (Plasencia, Spain), were involved in the study as control group. Signed informed consent was obtained from all subjects involved in the study. The study was conducted in accordance with the Declaration of Helsinki and approved by the Clinical Research Ethics Committee of Cáceres (Extremadura Health Service; reference: MASR/2016), and by the Bioethics and Biosecurity Committee (University of Extremadura; reference: 64/2016).

In the group of HT patients, CKD was established according to the KDIGO guideline.2 CKD was considered in the presence of albuminuria, albumin/creatinine ratio (A/C)≥30mg/g or an estimated glomerular filtration ratio (eGFR) using the equation CKD-Epidemiology Collaboration2 (CKD-EPI)<60ml/min/1.73m2.

HT patients were classified as G1 stage when eGFR was normal or elevated (≥90ml/min/1.73m2), G2 or slightly decreased eGFR (60–89ml/min/1.73m2), G3a or mild to moderately decreased eGFR (45–59ml/min/1.73m2), G3b or moderately to severely decreased eGFR (30–44ml/min/1.73m2), G4 or severely decreased eGFR (15–29ml/min/1.73m2) and G5 or renal failure (<15ml/min/1.73m2).

For HT patients who consented to participate in the study on a first visit, an interview was conducted in which their demographical and clinical data (history of diseases, concomitant diseases, drug treatment) were collected, and a general biochemistry analysis was performed, the eGFR was calculated, and a blood sample was taken from each participant for DNA extraction after the first visit. Later, according to the clinical requirements of each patient, they were cited 6, 12, 18 and 24 months. At each visit, a general biochemistry analysis was performed and the eGFR was calculated. In addition, cardiovascular events that occurred during the 24-month were also recorded.

DNA was isolated and purified from blood samples using QIAmp DNA extraction kit (Qiagen, Hilden, Germany). Genotyping for different CYPs and EPHX2 variant alleles was performed using a fluorescence-based allele-specific TaqMan (Thermo Fisher Scientific, Waltham, MA, USA) allelic discrimination assay (Table 1). PCR amplification for all single-nucleotide polymorphisms was performed in a PCR mixture consisting of 5μL of 2× Ex Taq Premix, 0.2μL of 50× Rox Reference Dye, 40× SNP Genotyping Assay Mix (Takara Bio Inc., Shiga, Japan) and 1μL of DNA (0.25ng/μL). Nuclease-free water was added to a final volume of 10μL. Amplification was carried out in an ABI 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The PCR conditions were an initial denaturation at 95°C for 30s, 40 cycles of 95°C for 5s and 60°C for 31s.

To assess for impaired kidney function, HT patients were categorized as progressors and non-progressors, defining progression as the decrease of eGFR≥10ml/min/1.73m2 in 24 months (≥5ml/min/1.73m2 per year).27

Logistic regression analysis (adjusted by age, gender, DM and dyslipidemia) was performed to assess the potential association between genotypes and impaired kidney function for 24 months in multiple inheritance models (dominant, recessive and additive). Odds ratios (OR) with corresponding 95% confidence intervals (95% CI), and the Hardy–Weinberg equilibrium (HWE) were determined using SNPStats software (www.snpstats.net).

Continuous variables were compared into different subgroups (eGFR stages, progression or genotypes) using Kruskal–Wallis test, Student's t-test or Mann–Whitney U test by SPSS statistical software (IBM SPSS Statistics 26; Chicago, IL, USA).

Fisher's exact tests were used to compare differences into variant allele frequencies between different groups (IBM SPSS Statistics 26; Chicago, IL, USA), and linkage disequilibrium (LD) statistics were obtained using Haploview software (www.broad.mit.edu/mpg/haploview). P-values lower than 0.05 were regarded as statistically significant.

Genotype frequencies both in HT patients (n=151) and in NT subjects (n=87) were in HWE. No significant differences in allele frequencies between HT patients (n=151) and in NT subjects for any of the SNP analyzed (Fig. 2), nor between the frequencies of the allelic variants in these two subpopulations and in the Spanish population (Fig. 2). In addition, after adjusting for age, gender, DM and dyslipidemia, there was no association with HT (data not shown), consequently, no association was found between any of the analyzed alleles and HT (Supplementary Table 1).

Fig. 2.

Frequencies (%) of minor allele frequencies (MAF) of CYPs and EPHX2 alleles in hypertensive patients (n=151), normotensive subjects (n=87) and in a Spanish population (n=107; IBS* population). *IBS: Iberian populations in Spain from 1000 Genome Project (http://www.ensembl.org/Homo_sapiens/).

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As shown in Fig. 3, the different SNPs of each EPHX2, CYP2C8/9, CYP4A11 and CYP2J2 gene loci were analyzed and all of them showed some type of LD between some of the SNPs evaluated, both in the group of HT patients and in the group of NT subjects, with the D′ values slightly higher in the group of NT subjects (Fig. 3).

Fig. 3.

Linkage disequilibrium plot of CYPs and EPHX2 variants alleles in normotensive subjects (n=87) and hypertensive patients (n=151). Linkage disequilibrium (LD) is displayed as pairwise D′ values. Shading represents the magnitude and significance of pairwise LD, with a red-to-white gradient reflecting higher-to-lower LD values. Dark red diamond without a number corresponds to D′ values of 1.0. (A) and (B) indicate the LD analysis of normotensive subjects (n=87) and hypertensive patients (n=151), respectively.

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The LDs with higher r2 values were the rs1123742 with rs3890011 variants of CYP4A11 (r2=0.80), rs11572325 with rs890293 variants of CYP2J2 (r2=0.82), and rs11572080 with rs10509681 (r2=0.97) and with rs1799853 (r2=0.87), and rs10509681 with rs1799853 (r2=0.89) of CYP2C8/2C9 locus.

To avoid a confounding factor on progression of decline renal function, patients in stage G5 (n=9), at the beginning of the study, were not included for this analysis. Regarding the allelic frequencies, the frequencies of the SNPs of CYP4A11 rs3890011 and rs9332982, as well as EPHX2 rs41507953 were significantly higher in the progressors (n=45) than in the non-progressor (n=97) group (P=0.036, P=0.026 and P=0.012, respectively; Fig. 4).

Fig. 4.

Frequencies (%) of minor allele frequencies (MAF) of CYPs and EPHX2 variants alleles in non-progressor (n=97) and progressor (n=45) patients (*P-value<0.05 by Fisher exact test).

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Using logistic regression, the ORs with 95% interval confidence (95% CI) for the different CYPs and EPHX2 genetic polymorphisms on the progression of impaired kidney function in HT patients (n=142), adjusted by age, gender, DM and dyslipidemia are shown in Table 3 for the dominant and recessive models. It should be noted that the CYP4A11 rs3890011, rs9332982 and EPHX2 rs41507953 polymorphisms, according to the dominant model, presented a high risk of impaired kidney function, with ORs (95% CI; P) of 2.07 (1.00–4.32; P=0.049), 3.02 (1.11–8.23; P=0.030) and 3.59 (1.37–9.41; P=0.009), respectively, and the EPHX2 rs1042032 polymorphism a greater risk according to the recessive model (OR=6.23; 95% CI=1.50–25.95; P=0.007). Instead, the CYP2C8 rs11572080 polymorphism presented a lower risk of progression than non-carriers of the T allele (OR=0.33; 95% CI=0.12–0.90; P=0.021).

Subsequently, the same analysis was also performed just in patients diagnosed with CKD (stages 3a, 3b and 4) at the beginning of the study (n=96). It was observed that adjusted ORs (age, gender, DM and dyslipidemia) were significant for the same polymorphisms as in the group of 142 HT patients but with higher ORs for all polymorphisms (Table 4).

The patients with diagnosis of dyslipidemia (n=90) presented higher frequencies of EPHX2 K55R (rs41507953) and *35A>G (rs1042032) variants than patients without dyslipidemia, 4% vs. 14% (P=0.005) and 16 vs. 27% (P=0.02), respectively. The ORs (95% CI; adjusted for age and gender) calculated for the presence of dyslipidemia were 4.08 (1.43–11.68; P=0.004) and 8.20 (1.01–66.37; P=0.04) to genotype EPHX2 (K55R) A/G-GG and EPHX2 *35 G/G carriers, respectively, while the OR (95% CI, adjusted for age and gender) for the EPHX2 (K55R) G – EPHX2 *35 G haplotype was 3.95 (1.40–11.10; P=0.01).

In contrast, none of the genetic variables analyzed was associated with CV events after two years of follow-up to HT patients that presented at least one CV event (n=40).

In the present study, it was initially evaluated the relationship between genetic polymorphisms of P450-derived eicosanoid genes and arterial HT, comparing a group of HT patients with a group of NT subjects. According to the results, not-significant differences were observed in the presence of some polymorphisms (Fig. 2). Furthermore, the frequencies found in both subgroups (HT patients and NT subjects) did not show significant differences with a Spanish control population (Fig. 2). These results do not agree with previous studies where the presence of the rs1126742 and rs9332982 polymorphisms of the CYP4A11 gene were associated with the development of essential HT in men30,31 and women,32 respectively, as well as with the Val433Leu variant (rs2108622) of the CYP4F2 gene in women.33 However, there are also studies in which no evidence was found of association between the risk of HT and the presence of these CYP4A1133,34 or CYP4F235 polymorphisms. Furthermore, the lack of association of CYP2C9, 2C8, 2J2 and EPHX2 polymorphisms with HT is consistent with that observed in previous studies.21,36–39 Therefore, the potential influence of these polymorphisms with HT is not clearly established. It may be partly due to the different groups of HT patients studied, differences in medication, concomitant diseases, lifestyle, etc. Furthermore, 20-HETE is excreted as a glucuronide conjugate and not in the free form and, in this sense, a study reported that the levels of 20-HETE glucuronide in the plasma of patients are higher than that found in the urine and that the fractional excretion is less than 1%.28 Therefore, urinary 20-HETE is not of renal origin and could simply reflects circulating levels of 20-HETE filtered and excreted.2

One of the most remarkable results of the study has been that CYP4A11 (rs3890011 and rs9332982) and EPHX2 (rs41507953 and rs1042032) polymorphisms were associated with increased progression of CKD. This fact could be explained by the hypothetical low level of EETs, which are associated with renoprotection and oppose the progression of CKD,14,17,20 since the polymorphism rs41507953 of EPHX2 codes for an enzyme with increased activity, so patients carrying this variant transform, at the renal level, more EETs towards their inactive derivatives DHETEs.

As for the other variant of EPHX2 gene associated with the progression of renal disease (rs1042032) is found in LD with the previous one (D′=1, Fig. 3), although its functional repercussion is unknown.

Regarding CYP4A11 variants associated with the progression of renal disease in this study (rs3890011, rs9332982), theirs functional repercussion in vivo (whether they increase or decrease the enzyme activity) is unknown. It is known that these polymorphisms have been associated in some studies with the risk of HT.30–32 However, at least one of them, the variant rs3890011, is in a high LD with the rs1126742 polymorphism (D′=1, r=0.80; P<0.0001; Fig. 3) which is associated with a loss of function in vivo,34 so indeed, 20-HETE levels will be low in progressor patients’ group. These results would contradict those found in a previous study where high levels of 20-HETE were related to a higher ORs of CKD progression.29 At could be possibly explained by the fact that 20-HETE concentrations were analyzed in plasma and not in urine in this study,29 which may be important as in a previous study they showed that 20-HETE conjugate levels in plasma are higher than those in urine.28 Furthermore, there are several studies supporting the hypothesis that low levels of 20-HETE at kidney might play an important role in the development of HT and renal injury, as well as in patients with inactive CYP4A11 mutations.17 This is supported by previous studies in which the formation of 20-HETE and EETs with fibrates was observed to reduce proteinuria and renal damage,40 as well as it has been reported that the 20-HETE excretion correlates with a decrease of eGFR in African American patients.28 Consequently, the results of the present study are consistent with the hypothesis that both EETs and 20-HETE oppose the development of CKD and the agents that increase intrarenal levels of EETs and 20-HETE, may protect against renal injury.17 This may be explained because reduced levels of 20-HETE and renal EETs in Dahl S rats promote sodium retention and the development of salt-sensitive HT, together with the decreased 20-HETE level also affects myogenic and tubuloglomerular feedback responses of the afferent arteriole, elevates glomerular capillary pressure, increases glomerular permeability to albumin, and up-regulates TGF-b expression, leading to the development of proteinuria and CKD.20,41,42

It should also be noted that, of the enzymes involved in the formation of CYPs-derived eicosanoids, the most expressed in the kidney are precisely these two enzymes, CYP4A11 and sEH. According to data provided by the open access resource for human proteins “The Human Protein Atlas”,43 the RNA expression in kidney of CYP4A11 and EPHX2 is 218.8 and 166.3nTPM (normalized transcript expression values), respectively. While the RNA expressions in kidney of CYP4F2, CYP2J2, CYP2C8 and CYP2C9 are much lower than the previous ones, being 40.9, 3.0, 4.3 and 1.4nTPM, respectively.

In contrast, the protective effect of progression of CYP2C8 mutation carriers is difficult to explain according to previous evidence, since it should be the progressor group who should present this polymorphism with a higher frequency than non-progressors since this polymorphism results in an enzyme with a decreased activity, which would cause low EET levels, which are related to renoprotection. However, it is an enzyme whose expression in the kidney is very low, practically insignificant (1.4nTPM), so its influence on renal function should not be as relevant as CYP4A11 or EPHX2 polymorphisms.

Finally, the association between rs41507953 and rs1042032 variants of EPHX2 gene and dyslipidemia was observed. A higher frequency of these variants was observed in patients with dyslipidemia compared to patients without dyslipidemia. Previous studies in animals suggest a role for the sEH enzyme and its gene EPHX2 in lipid regulation in response to diet. A study in mice fed with high-carbohydrate and high-fat (HCHF) diets, causing increased body weight, abdominal fat, and plasma lipid abnormalities, showed sEH enzyme activity in the liver 18% higher than in mice fed a standard diet.44 However, when rats fed a HCHF diet were administered an sEH inhibitor, the metabolic abnormalities were attenuated, suggesting a direct role of sEH in the aetiology of dyslipidemia in response to diet. On the other hand, total sEH activity was found to be higher in adipose tissue of rats fed a HCHF diet.45 In this sense, it has been well documented that a substantial proportion of malnourished individuals with anorexia nervosa have high serum cholesterol46 and that some variants in the EPHX2 gene were associated with total cholesterol levels in patients with anorexia nervosa.47

Regarding the relationship between the genetic variants analyzed in this study and CV events in HT patients, it was not observed in this study, similar to previous studies.36,37,39,48

Despite the limitations of the present study such as the number of participants, the lack of analysis of eicosanoid levels in both urine and/or plasma, as well as the potential influence of drug substrates of these enzymes on the outcome, the present results are the first to relate different polymorphisms of genes involved in eicosanoids metabolism to the progression of CKD in a group of patients over time (24 months).

Thus, further studies should be carried out focused on CYP4A11 and EPHX2 genes in a larger number of patients, analyzing levels of cytochrome P450-derived eicosanoids, primarily EETs and HETEs, in both urine and plasma and follow up patients over several years and taking into account treatments with drugs that are substrates or inhibitors of these cytochromes, such as antidiabetic, antihypertensive or NSAIDs drugs, which are widely used in this group of patients49 which can interfere with the formation of eicosanoids, especially EETs.

Moreover, and according to the results with the EPHX2 gene, the potential role of n−3 fatty acids, which could act as competitive substrates of CYPs and modify the synthesis of EETs and HETEs, should also be considered and monitored.

This is the first study in humans in which the relationship between the progression of impaired kidney function and different genetic polymorphisms involved in formation of eicosanoids, such CYP2 and CYP4 gene families, as well as EPHX2, has been evaluated. In this study has been found higher odds of CKD progression associated with CYP4A11 (rs3890011 and rs9332982) and EPHX2 (rs41507953 and rs1042032) polymorphisms, which may serve as biomarkers for improve clinical interventions aimed at avoiding or delaying, in CKD patients, progress to end-stage kidney disease needing dialysis or kidney transplant.