The Functional c.-2G>C Variant of the Mineralocorticoid Receptor Modulates Blood Pressure, Renin, and Aldosterone Levels
The mineralocorticoid receptor (MR) is essential in the regulation of volemia and blood pressure. Rare mutations in the MR gene cause type 1 pseudohypoaldosteronism and hypertension. In this study we characterized the common MR polymorphism c.-2G>C (rs2070951) in vitro and tested its influence on parameters related to blood pressure regulation and the renin-angiotensin system. In vitro studies showed that the G allele was associated with decreased MR protein levels and reduced transcriptional activation compared with the C allele. Association studies were performed with several outcome variables in 3 independent cohorts: a mild hypertensive group subjected to a salt-sensitivity test, a healthy normotensive group included in a crossover study to receive both a high and low Na/K diet, and a large cohort (The Netherlands Study of Depression and Anxiety), in which blood pressure was measured. Subjects with the GG genotype had significantly higher plasma renin levels both in the mild hypertensive group and in normal volunteers compared with homozygous C carriers. The GG genotype was also correlated with higher plasma aldosterone levels in healthy subjects. In both the mild hypertensive group and The Netherlands Study of Depression and Anxiety cohort the genotype GG was associated with higher systolic blood pressure in males. In conclusion, the G allele of the common functional genetic polymorphism c.-2G>C in the MR gene associates with increased activation of the renin-angiotensin-aldosterone axis and with increased blood pressure, probably related to decreased MR expression.
The mineralocorticoid receptor (MR) mediates aldosterone effects on electrolyte balance and blood pressure (BP). Classical MR-expressing tissues include the distal parts of the nephron, colon, salivary glands, and sweat glands, where MR regulates transepithelial sodium transport. However, MRs are also expressed in nonepithelial tissues, including the cardiovascular system and the central nervous system; in these tissues, glucocorticoids represent the predominant endogenous ligand.1 The MR belongs to the nuclear receptor superfamily and acts as a ligand-activated transcription factor regulating expression of a coordinate set of genes ultimately eliciting physiological aldosterone and cortisol responses. The gene coding for the human MR, NR3C2, is composed of 10 exons and spans over ≈400 kb. By means of alternative promoter use, alternative splicing, use of different translational start sites, and genetic polymorphisms, considerable variability in MR function has been observed.2,3
Sodium handling is highly variable between individuals, and genetic factors are involved in the development of hypertension.4 Rare mutations of the MR are responsible for mendelian disorders of renal salt handling associated with high or low BP. Loss of function mutations of the MR lead to type 1 pseudohypoaldosteronism,5,6 whereas the rare activating mutation S810L leads to juvenile hypertension exacerbated by pregnancy.7 Two recent studies showed associations between more common genetic variations, single nucleotide polymorphisms (SNPs), in the MR and BP.8,9 Previously we tested the amino acid changing SNP in exon 2, MRI180V (rs5522), in vitro and showed that the rs5522 G allele leads to a lower transactivational capacity.10 However, in a group of mild hypertensive individuals we did not find an association with BP and MRI180V,10 and the frequency of the same polymorphism was similar between hypertensive subjects and controls from a Brazilian birth cohort.11
MRc.-2G>C (rs2070951) is a frequent SNP located in the 5′-untranslated (UT) region of the NR3C2 gene, 2 nucleotides upstream of the first translation start site. The C allele of MRc.-2G>C has been associated with lower basal cortisol levels12 and a decrease in MR-dependent transcriptional activation in the presence of aldosterone.13 However MRc.-2G>C has not been tested for associations with salt handling, and the precise mechanism of action of this SNP is currently unclear.
In this study, we first investigated the functionality of MRc.-2G>C by testing its effect on MR protein expression, its influence on the MR-A/MR-B protein ratio, and the transactivational activity in vitro with different ligands. We then assessed the effect of this polymorphism on sodium handling and regulation of the renin-angiotensin system in 2-well phenotyped groups, a normotensive healthy group included in a crossover study to receive a low Na-high K or a high Na-low K diet and in a group of mildly hypertensive patients exposed to a salt sensitivity (Weinberger) test. Finally, we tested for an association between BP measures in a large multisite cohort for depression and anxiety.
Materials and Methods
Rabbit RCSV3 cells derived from a kidney cortical collecting duct14 (kindly provided by Prof P. Ronco, Hôpital Tenon, Paris, France) were grown as described previously15 and transfected using lipofectamine 2000 (Invitrogen) with 0.25 μg of pcDNA3 plasmid containing either MR-2G or MR-2C, 0.625 μg of a GRE2_TATA_luc reporter plasmid plasmid16 and 0.25 μg of pSVβgal. The day after transfection, steroids were added at different concentrations, and 48 hours after transfection, luciferase and β-galactosidase activities were assayed using the Dual-Light System and the Galacton-Plus Substrate (Applied Biosystems). Results were standardized for transfection efficiency and expressed as the ratio of luciferase activity over β-galactosidase activity in arbitrary units.
Recombinant plasmids used in this study are presented in Figure 1. For construction of recombinant plasmids and transactivation assays in COS-1 cells, please see the online Data Supplement at http://hyper.ahajournals.org.
Protein Expression Studies
For studies investigating the effect of the c.-2G>C SNP on protein synthesis, rabbit RCSV3 cells and COS-7 cells were seeded in 6-well plates at a density of 3×105 cells per well ≥6 hours before transfection in fresh medium without any added steroid. Cells were transfected by the calcium phosphate method with 0.66 μg of plasmids pcDNA3_1α2G-luc, pcDNA3_1α2C-luc, pcDNA3_1β2G-luc, or pcDNA3_1β2C-luc. Cotransfection of 0.16 μg of pSVβgal (Clontech) was performed to normalize for transfection efficiencies. Cellular extracts were assayed for luciferase and β-galactosidase activities as described previously.15 Results were expressed as the ratio of luciferase activity over β-galactosidase activity in arbitrary units.
For Western blot, Cos-1 cells were seeded in 6-well plates (Greiner Bio-One) at 2×105 cells per well. The cells were transfected the next day using Transit Cos transfection reagent (Mirus). Plasmids containing one of the MR variants, for example, MR-2G, MR-2C, mutated MR only expressing MR-A, mutated MR only expressing MR-B, or no MR (control), were used at 2 μg per well. Cells were harvested 48 hours after transfection. The Western blots using primary antibodies MR 1D5, detecting amino acid 1 to 18 and, therefore, only MR-A, and 2B7, detecting amino acid 64 to 82 and, therefore, both MR-A and MR-B, (a generous gift by C.E. Gomez-Sanchez, Division of Endocrinology, University of Mississippi, Jackson, MS) were performed as described previously17 The differences in intensity of the MR bands were quantified with ImageJ (National Institutes of Health, http://rsb.info.nih.gov/ij/).
RNA Isolation and Real-Time Quantitative PCR
For RNA isolation and real-time quantitative PCR procedures, please see the online Data Supplement.
In all of the studies, respondents provided written informed consent, and all of the studies were performed in accordance with the Declaration of Helsinki guidelines.
Mild Hypertensive Group
Ninety Italian white patients (34 women and 56 men; mean age: 46.0 years, mean body mass index: 26.8) with mild essential hypertension, that is, mean systolic BP (SBP) 152 mm Hg and mean diastolic BP 97.5 mm Hg were recruited by 9 medical centers. The patients were taken off antihypertensive medication 14 days before testing. After a normal sodium diet (150 mmol/d) for 3 days, patients were subjected to an acute salt-loading (constant rate intravenous infusion of 2 L of 0.9% NaCl carried out over 4 hours) and salt-depletion protocol (sodium restriction: 50 mmol plus 3 doses of 37.5 mg of furosemide) to evaluate the distribution of BP sensitivity to salt.18 If the difference between the mean arterial pressures at the end of the salt-loading and salt-depletion periods was greater than the median (10 mm Hg), the patient was classified as salt sensitive; otherwise, the patient was considered salt resistant. Twenty-four–hour urinary sodium excretion, upright plasma aldosterone (after 2 hours of orthostatism), and plasma renin activity were measured after 3 days of normal sodium diet (150 mmol/d) just before the salt load. Postload plasma aldosterone and renin activity were measured 4 hours after the beginning of the salt load. Urine electrolytes analyses, measurements of plasma renin activity, and plasma aldosterone concentration were performed as described previously.18
Forty healthy French white normotensive (BP <140/90 mm Hg in the supine position after 5 minutes of rest) men (18 to 35 years of age) were included in a crossover study to receive both a low Na+ (<20 mmol of NaCl per day) and high K+ (>140 mmol of KCl per day; low Na+-high K+ diet) diet or high Na+ (>250 mmol of NaCl per day) and low K+ (<50 mmol of KCl per day; high Na+-low K+ diet) diet for 1 week. The study design has been described previously in detail.19 Procedures were in accordance with institutional guidelines. Controlled Na+/K+ diet periods were separated by a 7-day washout period. On the ad libitum Na+ and K+ diet at baseline and on day 7 of each controlled Na+/K+ diet period, blood was sampled at 9:00 am in the fasting state after 1 hour of rest in the sitting position for plasma immunoreactive active and total renin and plasma aldosterone and atrial natriuretic peptide (ANP) determinations. Urine was collected in two 12-hour periods from 8:00 am to 8:00 pm and from 8:00 pm to 8:00 am and was used for hormone and electrolyte determinations. The methods used for collecting blood samples and for quantifying plasma active renin, total renin, ANP, and aldosterone were as described previously.19
Multisite Cohort for Depression and Anxiety
Data were obtained from The Netherlands Study of Depression and Anxiety, an 8-year longitudinal cohort study that includes 2981 Dutch white participants, aged 18 through 65 years. A detailed description of the study design and sample has been published previously.20 Netherlands Study of Depression and Anxiety is a multisite cohort study to describe the long-term course and consequences of depressive and anxiety disorders in which cardiological parameters, such as BP, were analyzed.20 Participants were recruited from different locations in The Netherlands (Amsterdam, Leiden, and Groningen). For the current study, data were used from the baseline interviews conducted between September 2004 and February 2007. Of the 2981 participants, 1860 subjects have been genotyped, and after quality control (described previously21), 1754 subjects (67.9% women; mean age: 42.35 years; SD: 12.49 years) were included in the study. The cohort consisted predominantly of subjects with current or remitted anxiety and/or depressive disorders at time of BP assessment. However, the presence of psychiatric diagnosis was not associated with BP.
BP was registered by the OMRON IntelliSense Professional Digital Blood Pressure Monitor, HEM-907XL (Omron Healthcare, Inc). SBP and diastolic BP were measured twice during supine rest on the right arm and were averaged over the two measurements. A correction was made for all of the individuals on hypertensive medication, which was considered as being used if subjects frequently (50% of days in last month) used antihypertensives (Anatomical Therapeutic Chemical [ATC] code C02), diuretics (ATC code C03), β-blocking agents (ATC code C07), or calcium channel blockers (ATC code C08). In accordance with earlier studies and based on the efficacy of antihypertensive drugs in randomized trials,22,23 we added 10 mm Hg to SBP and 5 mm Hg to diastolic BP for subjects who used antihypertensives.
Determination of Genotypes
For genotyping procedures, please see the online Data Supplement.
For detailed description of the statistical analyses, please see the online Data Supplement.
MRc.-2G>C Influences the Transactivation Activity of the MR
The influence of the MR-2G>C SNP on MR function was measured with an in vitro transactivation assay. RCSV3 and Cos-1 cells were transfected with plasmids containing either MR with the −2C nucleotide or MR with the −2G nucleotide (Figure 1). Dose-response curves in the presence of aldosterone or cortisol showed significantly higher transcriptional activity in the presence of the −2C allele than with the −2G variant in both cell models (RCSV3 cells: P<0.001 for both aldosterone [Figure 2, left] and cortisol [Figure 2, right]; Cos-1 cells: P<0.05 for aldosterone [Figure S1, left], P<0.001 for cortisol [Figure S1, right]; please see online Data Supplement).
MRc.-2G>C Affects Protein Expression Independent of the 5′-UT Region
In the human NR3C2 gene, two 5′-UT exons are alternatively transcribed and generate 2 different mRNAs coding for a unique MR protein.3 The c.-2G>C SNP is located in exon 2, 2 nucleotides upstream of the translation initiation site in the middle of the Kozak consensus sequence for translational initiation,24,25 which is highly conserved among the NR3C2 genes from several species (Figure 1A). We have investigated the influence of the c.-2G>C SNP on translational efficiency, in the context of both 5′-UT exons 1α and 1β. Chimeric constructs were generated, with exon 1α or 1β inserted together with the Kozak sequence containing either G or C immediately upstream of the coding sequence of the luciferase gene (Figure 1B). Transient transfections were performed in renal RCSV3 cells and in COS-7 cells, the amount of luciferase activity representing the amount of protein generated. In the presence of comparable mRNA levels (please see Figure S2A, available in the online Data Supplement), the C allele was associated with significantly higher protein levels compared with the G allele, both at 12 and 24 hours posttransfection in RCSV3 cells (P<0.0001; Figure 3A); this effect was observed in the presence of both the UT exons 1α or 1β. Interestingly, luciferase activity in the presence of the UT region 1β was ≈40% of that observed with exon 1α (P<0.0001). The same difference between the 2 alleles was observed in COS-7 cells (P<0.0001; Figure 3B), but only in the presence of the UT region 1α.
We then investigated whether the −2G>C SNP may affect alternative translation of the previously described human MR isoforms MR-A and MR-B, generated through the use of alternative translation initiation sites26 (Figure 1C). Expression vectors containing MR with either −2C or −2G and control constructs expressing only MR-A or MR-B were transiently transfected in Cos-1 cells. The MR mRNA levels after transfection were similar for MR-2C and MR-2G (Figure S2B). Two different primary antibodies were used in Western blots: 1D5 directed against amino acids 1 to 18 to visualize MR-A, whereas 2B7 (amino acids 64 to 82) detects both MR-A and -B (Figure 1C). Specificity of the antibodies was confirmed with the control constructs expressing only MR-A or only MR-B (Figure S3A and S3B). In Cos-1 cells transfected with MR-2G or MR-2C, only MR-A was expressed but not MR-B (Figure S3B). Western blots confirmed the differences in MR-A protein expression between −2G and −2C containing constructs (Figure S3C), MR-2C resulting in significantly increased MR-A expression (ratio of MR/tubulin: MR-2G, 0.300±0.014; MR-2C, 0.362±0.022).
Consequences of MRc.-2G>C on Renal Sodium Handling and BP Regulation
The MRc.-2G>C SNP was determined in subjects from 3 independent groups of patients. Allele and genotype frequencies were not significantly different among the 3 groups (χ22=5.36, P=0.07 and χ24=6.57, P=0.16, respectively; Table S1).
Mild Hypertensive Group
After 3 days of high-salt diet (150 mmol/d), plasma renin activity was significantly higher in mild hypertensive GG patients compared with the other genotypes (Table 1). In addition, the GG genotype was associated with significantly higher SBP levels in men but not in women (Table S2). No association of the MRc.-2G>C genotypes was found with other parameters tested in the cohort, such as 24-hour urinary sodium excretion and plasma aldosterone or plasma aldosterone and plasma renin activity levels after salt loading (P=0.42 and P=0.49, respectively). After the Weinberger test, the ratio of salt-sensitive/salt-resistant subjects was not significantly different among genotypes (χ21=1.69; P=0.43), and there was no relationship between the genotypes and BP response to the salt-sensitivity test (P=0.21; data not shown).
On the ad libitum Na+/K+ diet, 156 mmol of Na+ (interquartile range: 126 to 187 mmol) and 68 mmol of K+ (interquartile range: 59 to 81 mmol) were excreted in the urine in 24 hours. By controlling the Na+/K+ intakes, it was possible to achieve the desired Na+ and K+ balances, as reflected by 24-hour urinary NaCl and KCl excretion rates (Table 2). The 24-hour urinary Na+ and K+ excretion levels were identical for subjects of the 3 genotypes for all of the diets (Table 2).
Plasma active and total renin and aldosterone, as well as ANP, on the ad libitum Na+/K+ diet were within the physiological ranges and did not differ according to genotype (Table 2). As expected, plasma active and total renin and aldosterone concentrations increased with the low Na+-high K+ diet and decreased with the high Na+-low K+ diet (P<0.0001 between diets for all of the parameters). The changes in plasma ANP concentrations were in the opposite directions (Table 2; P<0.0001 between diets). On the high Na+-low K+ diet, GG subjects had significantly higher levels of plasma active and total renin and plasma aldosterone concentrations than CC subjects, with heterozygous GC subjects presenting intermediate values. A similar trend was observed on the low Na+-high K+ diet, but differences between GG and CC genotypes were not significant.
Netherlands Study of Depression and Anxiety Cohort
There was a significant association between SBP and MRc.-2G>C (P=0.041), even after adjustment for confounding factors (Table S3). GG subjects had significantly higher SBP than GC or CC subjects (mean SBP for GG: 138.2±1.9 mm Hg; GC: 137.3±1.8 mm Hg; CC: 135.2±1.9 mm Hg). Although we did not detect a sex×genotype interaction (P=0.36), we performed a separate analysis for both men and women. The association with MRc.-2G>C and SBP was significant for men but not for women (Table 3). Men with the GG genotype had significant higher systolic pressure (5.17 mm Hg) than those with the CC genotype (P=0.046). SBP among men was 147.0±21.2 mm Hg for the GG genotype, 143.8±17.6 mm Hg for the GC genotype, and 141.9±20.6 mm Hg for the CC genotype.
In this study we have undertaken the functional analysis of the c.-2G>C polymorphism, a frequent SNP in the NR3C2 gene coding for the MR, and its selective genotyping in subjects from different groups of subjects. The c.-2G>C variant was associated with differential expression of the MR in vitro; importantly, in vivo, this SNP influences circulating levels of plasma aldosterone and renin.
A possible role of the MRc.-2G>C polymorphism on translational efficiency had been suggested based on its location in the middle of the Kozak consensus sequence for translational initiation.24 First, we showed with 2 different endogenous ligands and in 2 different cell lines that MR translated from a construct carrying a C at position −2 was associated with a higher transcriptional response in vitro. These results are in contrast to previous work describing lower transactivation of a reporter gene by the −2C allele using aldosterone13; differences in methodology or the cell line used might explain this discrepancy. Second, using 3 different approaches and 2 different cell lines, including a kidney collecting duct epithelial cell line, we showed that the C allele results in more abundant protein expression than the G allele. Because mRNA expression is not modified by MRc.-2G>C, it is concluded that this polymorphism influences translation. This is in accordance with data showing that a C at position −2 is probably more favorable for translation: comparison of 1534 human transcripts has shown that the sequence surrounding the initiation codon contains a C at position −2 in 40% of cases, whereas a G nucleotide is present in only 18%.25 Furthermore, our results demonstrate that the 5′-UT has no influence on the observed effect of the SNP. In the presence of both UT exons 1α and 1β, the −2C allele was associated with higher protein translation. However, protein expression in the presence of the UT region 1β was ≈40% of that observed with exon 1α, indicating that the sequence of exon 1β is less optimal for translation. Finally, Western blot experiments showed that the MR-A isoform, translated from the first translation start, was more abundant in the presence of the C allele. However, transient transfection of MR containing either the −2 C or −2 G allele did not result in any detectable MR-B protein. As far as we know, only 1 study reported the MR-B isoform26; in that study MR-B was detected with an in vitro translation assay. Our results suggest that MR-B is not translated, at least not in Cos-1 cells. Given that the Kozak region preceding the translation start of MR-B is weak, the existence of MR-B in vivo needs further clarification. Taken together, we conclude that the C allele increases MR protein expression and, thereby, the in vitro transcriptional activity of the MR.
The physiopathological relevance of our in vitro results was tested by studying the association of the MRc.-2G>C polymorphism with parameters of BP regulation and electrolyte homeostasis in 2 groups of patients well phenotyped for BP and the renin-aldosterone axis under different experimental settings. Individuals carrying the CC genotype in either a healthy cohort under a high Na+-low K+ diet or a mild hypertensive cohort had significantly lower plasma renin concentration/renin activity levels, respectively. In parallel with the lower renin levels, the CC individuals in the healthy group also presented lower plasma aldosterone levels. The observed lower active renin, total renin, and lower aldosterone levels suggest a more efficient tubuloglomerular feedback in individuals with the CC genotype. According to the in vitro results obtained in this study, this effect might be attributed to more efficient sodium reabsorption because of higher levels of MR in the distal tubule, which was unmasked in conditions of low aldosterone synthesis and concentration, that is, on a high Na+-low K+ diet.27 There were trends in similar directions for these parameters in the healthy group at baseline or during the low Na+-high K+ diet, but this did not reach significance. The absence of significant difference between genotypes may be attributed to a low power of the study, which was not initially designed to test the effect of the MR polymorphism. Finally, both in the mild hypertensive group and in a large Dutch cohort, male GG carriers had a higher SBP. Taken together, these results indicate that the CC carriers may have a more favorable cardiovascular profile as compared with the GC and GG carriers.
Aldosterone has emerged as a key hormone determining cardiovascular and renal damage and risk prognosis, in addition to its role in BP regulation and potassium and sodium homeostasis. Within the last 10 years, blocking its effects with MR antagonists has been shown to have beneficial effects in congestive heart failure, especially after myocardial infarction, and proteinuric nephropathies.28,29 Although our results need to be replicated in larger cohorts well phenotyped for BP and the renin-angiotensin-aldosterone axis, our data suggest that functional variants of the MR may be associated not only with different cellular responses to aldosterone but also, indirectly, with increased aldosterone levels that may activate both genomic and nongenomic pathways in nonepithelial target tissues to promote deleterious cardiovascular effects.
Only male carriers of the MRc.-2G>C GG genotype showed higher BP compared with men with the other genotypes in the mild hypertensive cohort, the polymorphism not being associated with BP in women. The same sex-dependent association was found with SBP in the large-scale Dutch cohort, suggesting a sexual dimorphism. Many studies have reported sex-related differences in occurrence and severity of cardiovascular diseases related to the hormonal status.30 Interestingly, it has been shown that ovarian hormones positively affect salt sensitivity, protecting premenopausal women from the development of hypertension. After menopause, responsiveness of renin-angiotensin-aldosterone system changes, with a net increase in salt sensitivity.31 In addition, cortisol responses to stressors are different between men and women, and, as discussed above, cortisol might have an effect on epithelial MR as well.
Finding genetic variants involved in the regulation of BP offers mechanistic insights into the development of hypertension in the general population and helps in identifying novel targeted therapeutic strategies to prevent cardiovascular disease. Rare mutations of the NR3C2 gene result in monogenic diseases of sodium homeostasis and BP regulation. Our study provides evidence that frequent polymorphisms of the MR may exert quantitative effects on the activity of the renin-angiotensin-aldosterone axis and BP in the general population, modulating vulnerability for hypertension. Genotyping the common MR polymorphism c.-2G>C could help in identifying patients prone to develop hypertension and vascular disease, opening new strategies for prevention or targeted pharmacological treatment.
We thank Stephen B. Harrap for critical reading of the article and helpful comments.
Sources of Funding
M.C. has been a recipient of fellowships from the Fondation pour la Recherche Médicale, the Fondation Simone et Cino del Duca-Institut de France, and the University of Rome tor Vergata. This work was supported by a grant from the Fondation pour la Recherche Médicale and by institutional funding from Institut National de la Santé et de la Recherche Médicale, a grant from the Nederlandse Hersenstichting, Psychiatric Hospital Rivierduinen, and the Royal Netherlands Academy for Arts and Sciences. The infrastructure for The Netherlands Study of Depression and Anxiety is funded through the Geestkracht Program of The Netherlands Organization for Health Research and Development (Zon-MW, grant 10-000-1002) and is supported by participating universities and mental health care organizations (VU University Medical Center, GGZinGeest, Arkin, Leiden University Medical Center, GGZ Rivierduinen, University Medical Center Groningen, Lentis, GGZ Friesland, GGZ Drenthe, IQ Healthcare, Netherlands Institute for Health Services Research, and Netherlands Institute of Mental Health and Addiction [Trimbos]).
N.v.L. and M.C., C.B. and F.F., and R.H.D. and M.-C.Z. contributed equally to this work.
- Received May 3, 2010.
- Revision received May 21, 2010.
- Accepted August 26, 2010.
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