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Primary hypertension contributes to more than 50% of all the worldwide cardiovascular deaths. Current antihypertensive therapy is still targeting the physiological mechanisms regulating blood pressure, but not the “causal” ones. So far, genetic studies have been unable to prove gene causation in human primary hypertension. In this study we compared a standard antihypertensive losartan treatment with a pharmacogenomics-guided rostafuroxin treatment in never-treated Caucasian and Chinese patients with primary hypertension (PEARL-HT trial registration: EudraCT: 2010-022073-34, ClinicalTrials.gov: NCT01320397). Rostafuroxin is a digitoxigenin derivative that selectively disrupts the binding to the cSrc-SH2 domain of mutant α-adducin and of the ouabain-activated Na-K pump at 10 -11 M. Of 902 patients screened, 172 were enrolled in Italy and 107 in Taiwan. After stratification for country and genetic background, patients were randomized to rostafuroxin or losartan, being the difference in the fall in Office Systolic Blood Pressure (OSBP) after two-month treatment the primary endpoint. Three pharmacogenomic profiles (P) were examined, considering: P1, adding to the gene variants included in the subsequent P2, the variants detected by post-hoc analysis of a previous trial; P2, variants of genes encoding enzymes for endogenous ouabain (EO) synthesis ( LSS and HSD3B1 ), EO transport ( MDR1/ABCB1 ), adducin ( ADD1 and ADD3 ); P3, variants of the LSS gene only. In Caucasians, the group differences (rostafuroxin 50 μg minus losartan 50 mg in OSBP mmHg) were significant both in P2 adjusted for genetic heterogeneity (P2a) and P3 LSS rs2254524 AA [9.8 (0.6-19.0), P =0.038 and 13.4 (25.4-2.5), P =0.031, respectively]. In human H295R cells transfected with LSS A and LSS C variants, the EO production was greater in the former ( P =0.038); this difference was abolished by rostafuroxin at 10 -11 M. Chinese patients had a similar drop in OSBP to Caucasians with losartan but no change in OSBP with rostafuroxin. These results show that the selective blockade of mutant adducin and endogenous ouabain pressor mechanisms by rostafuroxin improves the therapy of primary hypertension in in Caucasians carriers of the related gene variants.

Among ~25% of patients with essential hypertension (EH), circulating endogenous ouabain (EO) and aldosterone (Aldo) are typically coelevated and their BP is especially salt-sensitive (SS). We probed for exaggerated natriuresis in a large cohort (712) of hypertensive patients that underwent an acute 2hr (T 120 ) load with saline (0.9%/2 L). Results: Using a quartiles analysis of sodium excretion, the 4th quartile subgroups showed higher SBP and DBP at T 120 , and at 2 hrs recovery (T 240 ). Fractional sodium excretion (FE Na) was significantly higher during loading and recovery in the 4 th quartile subgroup. Plasma Aldo was suppressed after saline in all subgroups as expected. SS patients showed an increased EO. In the 4 th quartiles EO was elevated at baseline and after saline (see figure), p Manova > 0.001 and the urinary Na/K ratio output was higher. Further, increases in plasma Na + were greater in the 4 th quartile group (1.91 ± 0.15 vs 0.83 ± 0.13 mM, p

Recently we have proposed LSS as genotype-based risk stratification to predict accelerated eGFR decay in a prospective cohort study of essential HYP patients. We extract clinical data of a general population from HYPERGENES consortium. We also collect genetic data for LSS polymorphism that was already demonstrated as involved in kidney damage. A cohort of 3137 patients were selected. At different age, as expected, HYP status have a deep impact on eGFR. Indeed in young (age < 50ys) eGFR is higher in HYP vs control (86.2 ± 19.3 vs 82.7 ± 16.3 ml/min; p=0.04); vice-versa in elder (age > 65ys) HYP have a reduction in eGFR (55.7 ± 12.9 vs 71.0 ± 12.9 ml/min; p Conclusion: Patients caring the risk allele of this specific LSS polymorphism seems to express hyper-filtration if compared to their counterparts.

Coronavirus disease 2019 (COVID-19) is a highly contagious viral disease causing a Severe Acute Respiratory distress syndrome. In 392 consecutive COVID-19 patients hospitalized in IRCCS San Raffaele in Milan (age 67 years range 28-94, Sex M, 74,7%) from march 2 until 25 April 2020, renal function was monitored. History of Hypertension (HYP) was the main comorbidity factors (58 %) under drug medications in 86.1% (ACEi/ARB 56,5). AKI (any stage) was detected in 6.2% of patients at emergency department (ED) admission. Baseline kidney function and HYP were the main determinants of ED-AKI (ED-AKI in HYP 89.5% vs 56.4 % in non-HYP; p=0.004) with an increased risk of 4.98 (95% CI 1.04-23.80; p=0.044) after adjustment by independent covariates, including age and respiratory distress.Moreover, MBP levels at presentation were inversely related with increased risk of ED-AKI. A stand-alone level of MBP < 86 mmHg (i.e.< 120/70 mmHg) or 65 yrs; 42.5% vs 24.3 %, X 2 16.62; p2 21.57; p

cGMP-dependent protein kinase type I is a major mediator of cGMP signaling in the cardiovascular system. Recent studies on cardiac-specific PRKG1 knockout mice demonstrated that cGMP-dependent protein kinase type I mediates the negative inotropic effect of cGMP in the myocardium. We therefore investigated the association between left ventricular (LV) function and common polymorphisms in the PRKG1 gene in a general population. In 609 randomly selected participants (51.2% women; mean age, 48.8 years; 36.6% hypertensive) who were free from overt cardiac disease, we performed echocardiography and genotyped intronic tag single-nucleotide polymorphisms (SNPs) rs1904694, rs7897633, and rs7905063 in PRKG1. On the basis of color Doppler myocardial motion data, we calculated end-systolic longitudinal and radial deformation (strain) of the LV inferolateral wall. In multivariable-adjusted analyses accounting for confounders and relatedness, systolic radial strain was significantly ( P ≤0.005) higher in homozygotes for rs1904694 (GG), rs7897633 (AA), and rs7905063 (TT) compared with heterozygotes or noncarriers. Haplotype analysis confirmed that LV radial strain was significantly higher in GAT homozygotes than in noncarriers (62.3% versus 56.0%; P =0.0005). Transmission of the PRKG1 GAT haplotype to informative offspring was associated with higher LV radial strain (effect size, 6.11%; P =0.017). For other LV phenotypes, none of the phenotype–genotype associations reached statistical significance. In conclusion, LV systolic radial function was associated with common polymorphisms in PRKG1 . If experimental studies and longitudinal follow-up of LV function confirm the causality of this association, interference with cGMP-dependent protein kinase type I function might be a target for pharmacological intervention.

A case–control study revealed association between hypertension and rs3918226 in the endothelial nitric oxide synthase ( eNOS ) gene promoter (minor/major allele, T/C allele). We aimed at substantiating these preliminary findings by target sequencing, cell experiments, and a population study. We sequenced the 140-kb genomic area encompassing the eNOS gene. In HeLa and HEK293T cells transfected with the eNOS promoter carrying either the T or the C allele, we quantified transcription by luciferase assay. In 2722 randomly recruited Europeans (53.0% women; mean age 40.1 years), we studied blood pressure change and incidence of hypertension in relation to rs3918226, using multivariable-adjusted models. Sequencing confirmed rs3918226, a binding site of E-twenty six transcription factors, as the single nucleotide polymorphism most closely associated with hypertension. In T compared with C transfected cells, eNOS promoter activity was from 20% to 40% ( P TT homozygotes and by 3.8/1.9 mm Hg in 2694 C allele carriers ( P ≤0.0004). The blood pressure rise was 5.9 mm Hg systolic (confidence interval [CI], 0.6–11.1; P =0.028) and 4.8 mm Hg diastolic (CI, 1.5–8.2; P =0.0046) greater in TT homozygotes, with no differences between the CT and CC genotypes ( P ≥0.90). Among 2013 participants normotensive at baseline, 692 (34.4%) developed hypertension. The hazard ratio and attributable risk associated with TT homozygosity were 2.04 (CI, 1.24–3.37; P =0.0054) and 51.0%, respectively. In conclusion, rs3918226 in the eNOS promoter tags a hypertension susceptibility locus, TT homozygosity being associated with lesser transcription and higher risk of hypertension.

To identify novel genes influencing blood pressure response to thiazide diuretic therapy for hypertension, we conducted genome-wide association meta-analyses of ≈1.1 million single-nucleotide polymorphisms in a combined sample of 424 European Americans with primary hypertension treated with hydrochlorothiazide from the Pharmacogenomic Evaluation of Antihypertensive Responses study (n=228) and the Genetic Epidemiology of Responses to Antihypertensive study (n=196). Polymorphisms associated with blood pressure response at P –5 were tested for replication of the associations in independent samples of hydrochlorothiazide-treated European hypertensives. The rs16960228 polymorphism in protein kinase C, α replicated for same-direction association with diastolic blood pressure response in the Nordic Diltiazem study (n=420) and the Genetics of Drug Responsiveness in Essential Hypertension study (n=206), and the combined 4-study meta-analysis P value achieved genome-wide significance ( P =3.3×10 −8 ). Systolic or diastolic blood pressure responses were consistently greater in carriers of the rs16960228 A allele than in GG homozygotes (>4/4 mm Hg) across study samples. The rs2273359 polymorphism in the GNAS-EDN3 region also replicated for same-direction association with systolic blood pressure response in the Nordic Diltiazem study, and the combined 3-study meta-analysis P value approached genome-wide significance ( P =5.5×10 −8 ). The findings document clinically important effects of genetic variation at novel loci on blood pressure response to a thiazide diuretic, which may be a basis for individualization of antihypertensive drug therapy and identification of new drug targets.

Abstract —Abnormalities in renal sodium reabsorption may be involved in the development and maintenance of experimental and clinical hypertension. Adducin polymorphism is thought to regulate ion transport in the renal tubule. It has recently been shown that there is a significant linkage of α-adducin locus to essential hypertension and that the 460Trp allele is associated with hypertension. Patients with this allele display larger blood pressure changes with body sodium variation. The aim of this study was to test whether α-adducin polymorphism is involved in abnormalities of renal function. Because proximal tubular reabsorption has been shown to be tightly coupled to renal perfusion pressure, this segmental tubular function was investigated in 54 (29 Gly/Gly and 25 Gly/Trp) untreated hypertensive patients in basal conditions with the use of endogenous lithium concentration and uric acid. Fractional excretions of lithium and uric acid were significantly decreased in the Gly/Trp hypertensive patients compared with the Gly/Gly hypertensives. The contribution of α-adducin to fractional excretion of lithium was investigated by multiple regression analysis. Adducin genotype was significantly ( R 2 =0.11, F=6.5; P + , urinary uric acid, mean blood pressure, and plasma renin activity were not related. In conclusion, the adducin gene can be considered to be a ‘renal hypertensive gene’ that modulates the capacity of tubular epithelial cells to transport Na + and hence contributes to the level of blood pressure.

The kidney plays an important role in salt and blood pressure (BP) homeostasis. In previous studies, variants in the genes for α-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.

Abstract The resolution of controversies that concern the detectability of an endogenous ouabain-like factor (OLF) in mammalian tissues and plasma was approached by the application of a standardized method for its extraction and quantification. Two independent assays were used to quantify the OLF: (1) a radioimmunoassay, which used a polyclonal antiouabain antiserum, and (2) a radioenzymatic assay based on the inhibition of dog kidney Na + ,K + -ATPase. Plasma and tissues were obtained from the Milan hypertensive strain (MHS) and the Milan normotensive strain (MNS) of rats and from healthy human volunteers. Results indicate that (1) a single high-performance liquid chromatography (HPLC) fraction identical to that of ouabain was identified by both assay methods in the rat hypothalamus and hypophysis and in both rat and human plasma; (2) dilution curves of OLF and standard ouabain were parallel and with a similar K d , both in radioimmunoassay (3 nmol/L) and ATPase assay (14 nmol/L); (3) after HPLC, OLF was similarly quantified by the two methods in the hypothalamus, hypophysis, adrenals, and plasma of rats and in human plasma; (4) OLF was present in larger amounts in the hypothalamus, hypophysis, and plasma of MHS rats than that of MNS rats; (5) the HPLC fraction of human plasma was quantified similarly by both assays (range, 60 to 150 pmol/L); (6) recovery of standard ouabain in pre-HPLC plasma extracts was approximately 90%; and (7) pre-HPLC OLF concentrations in human plasma ranged between 0.05 and 0.75 nmol/L. Rat cerebral tissues and both rat and human plasma contained measurable amounts of OLF, which were quantified similarly by radioimmunoassay and ATPase assay, both before and after HPLC fractionation. The increased MHS tissue and plasma levels of OLF are in keeping with the pathogenetic role of this factor in MHS hypertension.

To the Editor: Sodium-dependent mechanisms play a role in the pathogenesis of hypertension. He et al recently demonstrated that in normotensive and hypertensive subjects an acute reduction in salt intake, from 350 to 10 mmol/d for 5 days, was associated with a decline in serum sodium by ≈3 mmol/L.1 Conversely, a progressive increase in salt intake from 10 to 250 mmol/d by a daily amount of 50 mmol caused an increase in serum sodium in normotensive subjects, but not hypertensive patients.1 These investigators speculated that small changes in serum sodium might directly affect the hypothalamic control of blood pressure through the local pituitary renin-angiotensin system.1 Ouabain is a steroid hormone, which is released from the hypothalamus and the adrenal gland. It is implicated in sodium homeostasis and exerts direct actions on the vasculature, the heart,2 and tubular sodium reabsorption.3 In individuals randomly recruited from a Flemish population, blood pressure increased by 2.2 mm Hg systolic and 1.4 mm Hg diastolic for each 50-mmol/d increment in urinary sodium excretion when the plasma ouabain concentration was below the median (140 pmol/L).4 No association between blood pressure and urinary sodium was found when plasma ouabain exceeded the …

Genetics of hypertension with its disappointing results may evoke reactions ranging from enthusiasm and hope to suspicion and skepticism. A variety of reasons may account for these contrasting results1 that cannot be discussed here. However, in our opinion, 2 of these reasons are the most important ones: First, there is clear contradiction between the well established notion of the enormous heterogeneity of hypertension (in terms of molecular, biological, and clinical mechanisms) and the demand that a gene variant must be active across different populations, race, age, gender, etc. Second, the probability models of frequentist statistics, based on randomness, trivialize the ordered complexity of biological systems based on natural laws.2 In fact, all the available statistical genetic guidelines for defining causation of a single gene in a complex disease, like hypertension, are unable to fully account for genetic or environmental interactions.3 More than 50 years of pathophysiological, clinical, and epidemiological studies have clearly shown that these interactions are of paramount importance for the elevation of blood pressure. In fact, it is highly unlikely that the same molecular mechanism may account for a disease that affects up to 40% of the adult population of the industrialized countries. Interactions among these factor varieties can only be inferred from empirical observations and not from deductive mathematical logic.3,4 Along this line, studies trying to correlate genotypes with intermediate phenotypes, that are along biologically plausible pathways linking the genes of interest to blood pressure, may reduce this complexity and, therefore, provide more consistent data across the different studies.4 Intermediate phenotypes are also indispensable to dissect the heterogeneity of this human condition …