E. Brioni

Physiological Interaction Between α-Adducin and WNK1-NEDD4L Pathways on Sodium-Related Blood Pressure Regulation

The kidney plays an important role in salt and blood pressure (BP) homeostasis. In previous studies, variants in the genes for &agr;-adducin (ADD1), WNK1, and NEDD4L, which all regulate renal sodium absorption, have been associated with increased BP. However, findings have been inconsistent. We tested whether this is because of physiological interactions between the effects of variants in these genes. We assessed the single and combined effects of the ADD1 (Gly460Trp), WNK1 (rs880054 A/G), and NEDD4L (rs4149601 G/A) polymorphisms on renal and BP response to an acute Na load (n=344 subjects), BP decrease after 1 month of treatment with 12.5 mg of hydrochlorothiazide (n=193), and ambulatory 24-hour BP (n=690). Individually, the variants showed modest effects on some of the studied phenotypes. We found the ADD1 Trp allele to be permissive for the effects of variants of the other genes. In combination, the same variants (ADD1 Trp/WNK1 GG/Nedd4L GA+AA) showed a consistent effect on renal Na handling (P=0.009) and acute BP response to a saline infusion (P=0.021), BP lowering after thiazide treatment (P=0.008), and nocturnal systolic BP (P=0.044). Physiological interaction between the ADD1 and WNK1-NEDD4L pathways influences the effects of variants in these genes on sodium-related BP regulation. Relatively common alleles in the ADD1, WNK1, and NEDD4L genes when present in combination may have significant effects on renal sodium handling, BP, and antihypertensive response to thiazides.

Endogenous ouabain and the renin–angiotensin–aldosterone system: distinct effects on Na handling and blood pressure in human hypertension

Objective To evaluate whether the renin–angiotensin–aldosterone system (RAAS) and endogenous ouabain system differently affect renal Na handling and blood pressure. Methods Three hundred and one patients in whom we compared blood pressure, and renal Na tubular reabsorption in the basal condition and 2 h (T120) after saline infusion. Results Following multivariate-adjusted linear and quartiles analysis, baseline mean blood pressure (MBP) was significantly higher (113.7 ± 1.33 mmHg) in the fourth versus the first endogenous ouabain quartile (103.8 ± 1.04 mmHg) and the trend across the quartiles was highly significant (β = 0.23, P = 3.53e-04). In contrast, an inverse relationship was present in the renin activity (PRA) quartiles with MBP highest in the first (112.5 ± 1.26) and lowest in the fourth PRA quartile (107.6 ± 1.48, P = 0.039). Following an acute saline load, changes in MBP and the slope of the pressure–natriuresis relationship were inversely related across the PRA quartiles. The fractional excretion of sodium (FENa) showed a negative linear trend going from the first to the third endogenous ouabain quartiles (2.35 ± 0.17 and 1.90 ± 0.14%, P = 0.05). Patients in the fourth endogenous ouabain quartile (>323 pmol/l) showed increased FENa T120 (2.78 ± 0.18%, P < 0.01) and increased Na tubular rejection fraction (P = 0.007) after Na load. After the saline load, there was a biphasic relationship between plasma endogenous ouabain and FENa favoring Na retention at low endogenous ouabain and Na excretion at high endogenous ouabain levels. Conclusion The RAAS and endogenous ouabain system are two independent and complementary systems having an inverse (RAAS) or a direct (endogenous ouabain system) relationship with hemodynamic parameters.

cGMP-Dependent Protein Kinase 1 Polymorphisms Underlie Renal Sodium Handling Impairment

Defective pressure-natriuresis related to abnormalities in the natriuretic response has been associated with hypertension development. A major signaling pathway mediating pressure natriuresis involves the cGMP-dependent protein kinase 1 (PRKG1) that, once activated by Src kinase, inhibits renal Na+ reabsorption via a direct action on basolateral Na-K ATPase and luminal Na–H exchanger type 3, as shown in renal tubuli of animals. Because a clear implication of PRKG1 in humans is still lacking, here we addressed whether PRKG1 polymorphisms affect pressure-natriuresis in patients. Naive hypertensive patients (n=574), genotyped for PRKG1 rs1904694, rs7897633, and rs7905063 single nucleotide polymorphisms (SNPs), underwent an acute Na+ loading, and the slope of the pressure–natriuresis relationship between blood pressure and Na+ excretion was calculated. The underlying molecular mechanism was investigated by immunoblotting protein quantifications in human kidneys. The results demonstrate that the PRKG1 risk haplotype GAT (rs1904694, rs7897633, rs7905063, respectively) associates with a rightward shift of the pressure–natriuresis curve (0.017±0.004 &mgr;Eq/mm Hg per minute) compared with the ACC (0.0013±0.003 &mgr;Eq/mm Hg per minute; P=0.001). In human kidneys, a positive correlation of protein expression levels between PRKG1 and Src (r=0.83; P<0.001) or &agr;1 Na-K ATPase (r=0.557; P<0.01) and between &agr;1 Na-K ATPase and Na–H exchanger type 3 (r=0.584; P<0.01) or Src (r=0.691; P<0.001) was observed in patients carrying PRKG1 risk GAT (n=23) but not ACC (n=14) variants. A functional signaling complex among PRKG1, &agr;1 Na-K ATPase, and Src was shown by immunoprecipitation from human renal caveolae. These findings indicate that PRKG1 risk alleles associate with salt-sensitivity related to a loss of the inhibitory control of renal Na+ reabsorption, suggestive of a blunt pressure–natriuresis response.

Endogenous ouabain and aldosterone are coelevated in the circulation of patients with essential hypertension.

Objective: In the setting of normal sodium (Na) intake, many patients with hypertension have inappropriately elevated plasma aldosterone (Aldo) levels and may be at increased risk for tissue damage. Moreover, other adrenocortical steroids, including endogenous ouabain can stimulate tissue damage. As endogenous ouabain is often elevated in chronically Na-loaded states, is a vasoconstrictor, raises blood pressure (BP), and also promotes tissue fibrosis, we investigated the extent to which plasma Aldo and endogenous ouabain were coelevated among naïve hypertensive patients (NHP). We also investigated the impact of an acute salt load on these steroids, BP, and renal function. Methods: NHP (590) were grouped in tertiles based on their baseline plasma Aldo (mean ± SEM first 7.59 ± 0.18, versus third 24.15 ± 0.31 ng/dl). Baseline plasma renin activity (2.4 ± 0.1 versus 1.2 ± 0.1 ng/ml per h, P < 0.001), endogenous ouabain (268 ± 14.9 pmol/l versus 239.0 ± 13.6 pmol, P < 0.01) and DBP (91.9 ± 0.76 versus 89.6 ± 0.71 mmHg, P = 0.017) were higher in NHP in the third versus the first Aldo tertile, respectively. Results: Acute Na loading showed that the BP of the third Aldo tertile NHP was especially salt-sensitive (slope of pressure–natriuresis relationship 0.015 ± 0.002 versus 0.003 ± 0.001 &mgr;Eq/mmHg per min, P = 0.00024 after adjustment for sex, BMI, and age). Regression analyses showed that plasma Aldo and endogenous ouabain were linearly related (&bgr; = 0.181, P = 0.0003). Conclusion: Among patients with essential hypertension, circulating endogenous ouabain and Aldo are typically coelevated and their BP is salt-sensitive. In conditions where Aldo is inappropriately elevated, both Aldo and endogenous ouabain may contribute to adverse cardiovascular and renal outcomes.

Lanosterol Synthase Gene Polymorphisms and Changes in Endogenous Ouabain in the Response to Low Sodium Intake

Circulating levels of endogenous ouabain (EO), a vasopressor hormone of adrenocortical origin, are increased by sodium depletion. Furthermore, lanosterol synthase, an enzyme involved in cholesterol biosynthesis, has a missense polymorphism (rs2254524 V642L) that affects EO biosynthesis in adrenocortical cells. Here, we investigated the hypothesis that lanosterol synthase rs2254524 alleles in vivo impact the blood pressure (BP) and EO responses evoked by a low dietary Na intake (<100 mEq/d, 2 weeks) among patients with mild essential hypertension. During the low salt diet, the declines in both systolic BP (SBP: −8.7±1.7 versus −3.0±1.5; P=0.013) and diastolic BP (DBP: −5.1±0.98 versus −1.4±0.94 mm Hg; P<0.05), and the slope of the long-term pressure–natriuresis relationship affected significantly the presence of the lanosterol synthase rs2254524 A variant (AA: 0.71±0.22, AC 0.09±0.13, and CC 0.04±0.11 mEq/mm Hg/24 h; P=0.028). In addition, BP rose in ≈25% of the patients in response to the low salt diet and this was associated with increased circulating EO. Lanosterol synthase gene polymorphisms influence both the salt sensitivity of BP and changes in circulating EO in response to a low salt diet. The response of BP and EO to the low salt diet is markedly heterogeneous. Approximately 25% of patients experienced adverse effects, that is, increased BP and EO when salt intake was reduced and may be at increased long-term risk. The augmented response of EO to the low salt diet further supports the view that adrenocortical function is abnormal in some essential hypertensives.

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.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.

ENDOGENOUS OUABAIN: A HORMONAL EXPLANATION OF THE VARIABILITY OF THE RESPONSE TO LOW-SODIUM DIET IN ESSENTIAL HYPERTENSION: PP.23.419

Objective: European Guidelines suggest behavioural and farmaceutical treatment of hypertension, considering dietary Na restriction as milestone of the behavioural treatment. Epidemiological studies showed great BP variability in response to dietary Na restriction. We investigate the role of contro-regulatory hormones as plasma renin activity (PRA), plasma Aldosterone (ALDO) and endogenous ouabain (EO) in determining BP response. Design and Method: 271 never treated essential hypertensive patients were included in the study and received the low-sodium diet (100 mEq/day dietary sodium intake) for one month. We measured dietary compliance by 24 hours urinary Na excretion measurement at each visit. At each visit we measured BP and we performed blood and urinary test to evaluate renal function, PRA, aldosteron and EO. Results: The dietary compliance was 47% after 15 days and 38% after one month. After low-sodium diet BP fall is greater in compliant patients compared to non compliant ones (Compliant SBP/DBP decrease −9,01/−4,97 mmHg; Non compliant SBP/DBP decrease −4,24/−2,23 mmHg; p = 0,007 for SBP and p = 0,031 for DBP). In 30% of compliant patients BP increased after low Na intake (SR): this paradoxical response is associated with a consensual increased EO (+26,9 pM/L in SR pts vs −1,22 pM/L ANOVA p = 0,025). However, a negative correlation between the variation of DBP and the variation of plasma Na (r = −0,237; p = 0,047) was present. The increases in PRA and ALDO were inversely related to urinary Na (r = −0,211, p 0,03 and r = −0,346, p = 0.001, respectively), but not with BP response. Conclusions: 1. Renin-aldosteron system is the major regulatory system to prevent the excessive volume depletion after low-sodium diet. 2. Endogenous Ouabain seems to regulate BP variability in those patients that modified plasma Na concentration, after reduction of dietary Na intake. 3. Increase in plasma EO may explain the paradoxical BP response. Figure 1. No caption available.