Paolo Manunta

CA-Repeat Polymorphism in Intron 1 of HSD11B2: Effects on Gene Expression and Salt Sensitivity

Mutations in the HSD11B2 gene encoding the kidney (11-HSD2) isozyme of 11beta-hydroxysteroid dehydrogenase cause apparent mineralocorticoid excess, a form of familial hypertension. Because the hypertension associated with AME is of the salt-sensitive type, it seemed possible that decreases in 11-HSD2 activity might be associated with salt sensitivity. To examine this, Italians with mild hypertension underwent a protocol consisting of a rapid intravenous saline infusion and subsequent furosemide diuresis. To determine whether there were genetic associations between HSD11B2 and salt sensitivity, 198 Italians were genotyped for a CA repeat polymorphism (11 alleles) in the first intron. Increased differences in mean arterial pressure between the sodium loaded and depleted states were correlated with shorter CA repeat length (R=0.214, P=0. 0025). The effect behaved as a recessive trait. This suggested that decreased HSD11B2 expression was associated with shorter CA repeat length. Furthermore, activity of renal 11-HSD2 as measured by an increase in the ratio of urinary-free cortisol/urinary-free cortisone was lower in 33 salt-sensitive subjects (urinary-free cortisol/urinary-free cortisone 0.89+/-0.04 [mean+/-SE]) compared with 34 salt-resistant subjects (0.71+/-0.04, P<0.001). However, when minigenes containing either 14 or 23 CA repeats were transfected into rabbit or human kidney cortical collecting duct cells, the construct with 14 repeats was instead expressed at levels 50% higher than those of the construct with 23 repeats, as determined by reverse transcription-polymerase chain reaction. We conclude that polymorphisms in HSD11B2 and decreased 11-HSD2 activity are associated with sensitivity to sodium loading, but a functional explanation for these associations remains to be elucidated.

Endogenous ouabain and hemodynamic and left ventricular geometric patterns in essential hypertension

We sought to evaluate the relationships among circulating levels of an endogenous ouabain-like factor (EO) and systemic hemodynamics and left ventricular (LV) geometry in patients with recently diagnosed essential hypertension. We selected 92 never-treated patients with essential hypertension. Blood samples were drawn for estimation of plasma EO (radioimmunoassay) and subjects underwent echocardiographic examination to evaluate LV end-systolic and end-diastolic wall thickness and internal dimensions. LV volumes, stroke volume, cardiac output, total peripheral resistance, LV mass, and relative wall thickness were calculated, and all except the last parameter were indexed by body surface area. LV mass also was indexed by height. On the basis of the values of LV mass index (body surface area or height) and relative wall thickness, subjects were divided into groups with either normal geometry, concentric remodeling, concentric hypertrophy, or eccentric nondilated hypertrophy. In the study population as a whole, circulating EO levels were significantly and directly correlated with mean blood pressure (r = 0.21, P = .048), relative wall thickness (r = 0.34, P = .001), and total peripheral resistance index (r = 0.37, P = .0003). Plasma EO also was significantly and inversely correlated with LV end-diastolic volume index (r = -0.32, P = .002), stroke index (r = -0.34, P = .0009), and cardiac index (r = -0.35, P = .0007). In multiple regression analysis, plasma EO was an independent correlate of total peripheral resistance index, cardiac index, and relative wall thickness. Regardless of the indexation method used for LV mass, plasma EO was higher in patients with concentric remodeling than in those with either normal geometry or concentric hypertrophy. Plasma EO tended to be higher (indexation by body surface area) or was significantly higher (indexation by height) in subjects with concentric remodeling than in those with eccentric nondilated hypertrophy. Patients with concentric remodeling showed the highest total peripheral resistance index and the lowest cardiac index. Our data suggest that EO plays a role in regulating systemic hemodynamics and LV geometry in patients with essential hypertension.

ADDUCIN IN ESSENTIAL HYPERTENSION

However adducin polymorphisms account for only a portion of hypertension both in humans and rats. Therefore additive or epistatic interactions with other genes involved in renal sodium handling need to be studied.

6C.06: GENES INVOLVED IN BLOOD PRESSURE RESPONSE TO ACUTE AND CHRONIC SALT MODIFICATIONS: IDENTIFICATION OF A NEW PATHWAY.

Objective: A moderate reduction in salt intake reduces BP in most but not all individuals. Identification of genetic loci combination is a difficult task. Recently, the uromodulin (UMOD) gene has been associated with renal damage and hypertension. Design and method: Present study evaluated the role Endogenous Ouabain (EO), polymorphisms in a candidate gene related to its synthesis, Lanosterol Synthase (LSS), and in UMOD in variability of response to acute and chronic body Na variations. Acute Na load protocol in 608 naïve hypertensive patients (NHP) was carried out. 183 NHP followed a low Na intake diet for 15 days. UMOD and LSS gene polymorphisms have been tested. Results: Acute protocol: NHP carrying UMOD GG/LSS AA display a pressure-natriuresis curve with negative slope (Salt Resistant), while those with UMOD AA/LSS AA showed a right shift of pressure-natriuresis curve (Salt Sensitive, upper panel). Low Na intake: In a dietary compliant group with the reduction of UNa excretion, a direct (&bgr;=0.213) relationship (p = 0.026) between the change in EO and BP was found. When LSS and UMOD gene variants were analyzed together, a significant interaction was detected: those patients homozygous for the A alleles of both gene variants displayed a 5-fold greater decline in SBP than patients carrying other allele combinations (lower panel). Figure. No caption available. Conclusions: We identify a genetic interaction that characterized a subgroup of patients. In this pathway UMOD may affect renal tubular Na excretions, whereas LSS affects vasoconstrictor activity modulating circulating EO levels. This new pathway is relevant for blood pressure response during both acute and chronic salt modification.

ALPHA-ADDUCIN POLYMORPHISMʼS INFLUENCE ON ISCHEMIC STROKES

Objective: rs4961 Gly460Trp variant of the alpha adducin gene (ADD1) has been associated with renal sodium retention and salt sensitive hypertension. Previous studies indicated that carriers of the 460Trp allele have a higher risk of hypertension and cardiovascular (CV) diseases compared to wild-type homozygotes (Gly460Gly). The aim of this study is to assess whether there is a correlation between this ADD1 variant and the development of ischemic strokes in an Italian population. Design and method: 212 patients with ischemic stroke (IS) were recruited from the Stroke Unit of San Raffaele Hospital in Milan and divided into four categories according to Oxford Classification. These patients were compared to a cohort of elder general population (EGP, 128 patients) and a cohort of hypertensive patients (HYP, 2404 patients), both with no history of strokes. All patients analysed were genotyped for adducin family (ADD1, ADD2, ADD3) and other hypertension-related genes. Scientific data: in IS group mean age at strokes’ diagnosis was 72.34 ± 11.9, 61% men, 39% women. The incidence of CV risk factors was: hypertension 66%, diabetes 22%, hypercholesterolemia 40% and hypertriglyceridemia 13%, previous stroke 14%. Mean creatinine value was 1.07 ± 0.66 mg/dL. Two comparable populations were selected with similar age (71.28 ± 6.9 years for EGP) and with the same incidence of risk factors (for HYP population). Notably, the presence of subjects homozygous for ADD1 mutated allele (rs4961 TrpTrp) is more than double in patients bearing ischemic stroke than in the two other (6.1% IS vs. 2.3% EP, p = 0.049; 6.1% IS vs. 2.2 HYP, p = 0.009; fig. 1). There was no statistically difference among the various types of stroke. No other significant associations were identified with other gene variations. Results: These data suggest a correlation between mutated alpha-Adducin and the increased incidence of all types of ischemic stroke. No correlation was found with the other hypertension-related genes analysed. Conclusions: rs4961 alpha-Adducin polymorphism should be considered as an independent risk factor for ischemic strokes. Furthermore, ischemic stroke does not appear directly linked to hypertension. Finally, alpha-Adducin polymorphism itself might be a candidate gene in the pathogenesis of stroke. Figure. No caption available.

HYPERTENSION IN HIGH SCHOOL STUDENTS: GENETIC AND ENVIRONMENTAL FACTORS (HYGEF STUDY)

Objective: To evaluate the impact of gene pathways (ADDs, Endogenous Ouabain genetic polymorphisms) in the transition from normotension to hypertension HT we perform a large epidemiological study in 3 regions of Italy (Milan, Mi, Lombardy, Livorno, Li, Tuscani, and Grottaglie, Gt, Puglia) among young (age < 18) high school students. Design and method: During two consecutive medical visits, we collected the anthropometric data and blood pressure values and a spot urine, saliva sample was taken for the DNA study. Results: Preliminary results obtained on 2,635 boys (f 1,501, m 1,134, age 16,80 ± 1,83 years) show a regional difference both for estimated urinary Na+ (Li 171.5 ± 2.1, Mi 178.7 ± 2.8, Pt. 196.4 ± 2.5 mEq/24 h, p < 0.001), although SBP values were higher in Tuscany (Li 121.1 ± 0.36, vs Mi 118.9 ± 0.4, Gt 118.4 ± 0.41 mmHg). SBP throughout the sample was significantly correlated with BMI (r = 0.324, p < 0.0001), and with Ur.Na+ (r = 0.138, p < 0.0001). In the analysis of the genotypes, removed environmental factors, the Lanosterol Synthasi (LSS) polymorphism, the enzyme involved in the synthesis of Endogenous Ouabain (EO) and cholesterol, was associated with increased DBP values (LSS AA 69.2 ± 0, 67, LSS AC 68.3 ± 0.32, LSS CC 66.7 ± 0.3 mmHG, p < 0.0001). Carriers of both mutated variants of ADD1 and ADD2 (ADD1GT / ADD2 CT, n = 167) showed greater (p = 0.015) urinary excretion (192.9 ± 4.26 mEq / 24 h) than subjects with genetic variants ADD1 GG / ADD2 CC (n = 826) (185.2 ± 1.9 mEq / 24 h). The Na/K urinary ratio was increased in the boys carrying LSS AA/CYP1A1 CC 4.04 ± 0.41 vs. LSS GG/CYP1A1 AA 3.19 ± 0.35 mEq / L, suggesting an interaction between EO and aldosterone. Conclusions: The results obtained in this young population confirm the role of the Adducin-EO genetic network and allow to identify interactions between environmental factors (different eating habits) and the genetic polymorphisms linked to hypertension. RF-funded study: PE-2011-02346988

GENE INTERACTIONS OF ALPHA-ADDUCIN AND LANOSTEROL SYNTHASE IMPACT RENAL IMPAIRMENT IN SALT SENSITIVE HYPERTENSION

Objective: The genes involved in the development of renal damage and salt sensitive hypertension (SS) are unknown. Renal impairment is considered to be the major determinant of salt sensitive hypertension. Design and method: We explored the roles of alpha-adducin (ADD1) and lanosterol synthase (LSS), genes coding for structural proteins of the cell membrane and Endogenous Ouabain (EO), respectively, on renal Na handling and their genetic interactions in a large cohort of naïve hypertensive patients (NHP) whose salt sensitive phenotype was characterized. Acute saline load (NaCl 308 mEq/2 h e.v.) was performed in 774 NHP (age 44.95 ± 9.61 years), and functional and hormone renal parameters were tested. Results: Under baseline conditions NHP carriers of the LSS AA genotype showed lower GFR (n = 68, 115.7 ± 4.6 ml/min) compared to LSS C carriers (124.6 ± 1.4 ml/min, p = 0.066). After an acute saline test, GFR increased in both groups, while the urinary volume and urinary creatinine were lower in LSS AA. Analysis of gene*gene interactions demonstrated that NHPs carrying both mutated GT ADD1 and homozygous for LSS C variants excreted the sodium load more rapidly than their ADD1*LSS mutated variants. On the other hand, an additive (p = 0.003) effect of ADD1 GT & LSS AA variants on the pressure-natriuresis relationship was observed, with a significant right shift along the x axis. Finally, circulating EO was modified according to the LSS variant: in SS LSS AA, EO rose signicantly during the recovery while in SR LSS AA, EO was suppressed as were aldosterone and PRA. Conclusions: The results demonstrate: 1. NHPs carrying the LSS AA genotype have reduced GFR under basal conditions; 2. Salt Sensitive LSS AA are EO non-modulators, since their circulating EO does not decline under circumstances where the RAAS is suppressed; 3. The ADD1*LSS interaction identifies those NHPs with impaired renal Na handling.

[OP.2A.05] LANOSTEROL SYNTHASE GENE POLYMORPHISMS IMPACT THE DECLINE IN RENAL FUNCTION AMONG HYPERTENSIVE PATIENTS

Objective: Cholesterol is an essential component of mammalian cell membranes and serves as a precursor for bile acids and various steroid hormones. Lanosterol, the first committed intermediate in cholesterol biosynthesis, is coded by the Lanosterol Synthasis gene (LSS) with a missense polymorphism that affects EO biosynthesis in adrenocortical cells. Recently, we reported that the LSS AA genotype is associated with salt-sensitive hypertension. Exposure to increased circulating EO causes glomerular damage, and is a risk factor for acute kidney injury. In this report, we explore the importance of LSS in the progression of chronic kidney disease (CKD). Design and method: A cohort of 338 naïve hypertensive patients (f 162, m 176, age 42.7 8.41 years), were enrolled in a prospective follow-up study (5.32 ± 4.37 years) in which blood pressure values were kept at goal with ACEi plus diuretic and, when needed, a Ca2+ channel antagonist. Results: Blood pressure values (SBP/DBP) after 4.6 years of follow-up were at target for all patients with no difference according to genetic polymorphism (LSS AA 138/85 AC 137/87 CC 137/87, respectively). The slope in eGFR (CKD-EPI) was 1.43 ± 0.47 ml/1.73 m2/yr in the whole studied population. When analysed according to the LSS genotype, the decline in renal function was double in the AA homozygotes (LSS AA -2.01 ± 2.4 vs CC 2.23 ± 1.20 ml/1.73 m2/yr; p = 0.024). The impact of such LSS polymorphism was also reflected in renal survival analysis (p = 0.001). Figure. No caption available. Conclusions: Our findings further support the role of LSS polymorphisms in the progression of CKD. This metabolic pathway may accelerate the decline in renal function via its effects on glomerular podocyte and tubular components.

[OP.7C.03] KLOTHO GENE POLYMORPHISM INTERACTS WITH ADDUCIN-ENDOGENOUS OUABAIN-NA+-K+ ATPASE SYSTEM IN SALT SENSITIVE HYPERTENSION

Objective: The genetic basis of salt sensitive (SSH) hypertension consists in a very complex network of dynamic interaction among genetic-environmental factors that may change with aging. The existence of a regulatory genetic network (RGN) as triggering mechanism of SSH has been already postulated for Adducin-Endogenous Ouabain-Na+-K+ ATPase (ADD-EO-NKA) system. The observation that Klotho (KL) is a newly discovered aging suppressor gene can directly affect the Na+, K+-ATPase activity opening new possible interaction with the RGN. Aim of this study is to evaluate the interactions between Add-EO-NKA and KL genes on pressure-natriuresis relationship in SSH. Design and method: A large cohort of 655 naive hypertensive patients underwent acute Na Load test for phenotyping the pressure natriuresis relationship (PNat). Results: Univariate analysis showed significant interactions between Klotho G/T SNP and RGN network (SNPs in loci of ADDs, EO synthesis, metabolism, and activity) with PNat slope. Figure. No caption available. Furthermore, those patients carrying together mutate KL and NCX1 (SLC8A1) variants showed a rightward shit of PNat curve than in wild type condition. Similar differences are present in delta systolic BP (8.92 vs 2.26 mmHg), with lower fractional excretion of Na (FE Na 1.48 vs 2.40 %), and increased circulating EO (251.41 vs 190.75 pM) after Na load. Conclusions: Klotho beside its effects on Ca/P metabolism may influence renal sodium handling and vascular activity through different component of EO-ADD-NKA system.

[PP.06.05] RELATIONSHIP BETWEEN URIC ACID AND BLOOD PRESSURE LEVELS IN GENERAL POPULATION

Objective: Elevated plasmatic levels of uric acid (UA) were frequently associate with hypertension however its role in hypertension pathogenesis is unclear. Design and method: Our aim is to investigate the role of UA on blood pressure (BP) and its genetic determinates on a general population (not treated) of 1350 patients undergoing 24 h ambulatory BP monitoring (AMBP). Results: As expedited, we found of correlation between systolic and diastolic mean 24 h BP values with age (p < 0.001), sex (p = 0.004), BMI (p < 0.001) and plasmatic levels of creatinine (p = 0.023). We also find a strong correlation of BP with UA alone (p = 0.001). Only age and UA remain significant in multivariate analysis with all elements under investigation (respectively p = 0.01 and p = 0.04). We found that UA plasmatic levels are associated with sex (multivariate analysis: p < 0.001), BMI (p = 0.004), renal function (p = 0.001) and with genetic polymorphisms of Protein Kinase CGMP-Dependent Type I (PRKG) and lanosterol synthase (LSS) genes (respectively p = 0.025 and p = 0.05 after correction for clinical covariates included sex, BMI and creatinine level). Conclusions: Our data confirm that UA levels are a strong and independent determinant of BP (both systolic and diastolic) in the general population. Moreover it seems to be an independent element of metabolic syndrome. Finally, UA plasmatic levels are strictly associated with specific clinical and genetic characteristic. In particular we identify two new genes that could play a substantial role in determination of UA plasmatic level.