Lorena Citterio

Common noncoding UMOD gene variants induce salt-sensitive hypertension and kidney damage by increasing uromodulin expression

Hypertension and chronic kidney disease (CKD) are complex traits representing major global health problems. Multiple genome-wide association studies have identified common variants in the promoter of the UMOD gene, which encodes uromodulin, the major protein secreted in normal urine, that cause independent susceptibility to CKD and hypertension. Despite compelling genetic evidence for the association between UMOD risk variants and disease susceptibility in the general population, the underlying biological mechanism is not understood. Here, we demonstrate that UMOD risk variants increased UMOD expression in vitro and in vivo. Uromodulin overexpression in transgenic mice led to salt-sensitive hypertension and to the presence of age-dependent renal lesions similar to those observed in elderly individuals homozygous for UMOD promoter risk variants. The link between uromodulin and hypertension is due to activation of the renal sodium cotransporter NKCC2. We demonstrated the relevance of this mechanism in humans by showing that pharmacological inhibition of NKCC2 was more effective in lowering blood pressure in hypertensive patients who are homozygous for UMOD promoter risk variants than in other hypertensive patients. Our findings link genetic susceptibility to hypertension and CKD to the level of uromodulin expression and uromodulins effect on salt reabsorption in the kidney. These findings point to uromodulin as a therapeutic target for lowering blood pressure and preserving renal function.

Adducin- and Ouabain-Related Gene Variants Predict the Antihypertensive Activity of Rostafuroxin, Part 2: Clinical Studies

Five genetic variants that affect Na,K-ATPase interactions predict the blood pressure response to rostafuroxin but not to losartan and hydrochlorothiazide. Help for Hypertension As if changing its mind about how best to detoxify the body, the kidney first secretes a filtrate that contains almost everything in the blood but then recaptures much of it by pumping essential water, salts, and other molecules back in. The Na+, K+-ATPase, or sodium pump, recaptures sodium salts, and because Na+ is the prime determinant of extracellular fluid volume in the body, regulation of this pump controls blood pressure. Now a pair of papers describes how an antihypertension drug can correct abnormal sodium pumping and how this understanding of the drug’s mechanism points to a genetic signature that can predict whether a patient will respond to the drug. One cause of hypertension is a particular variant(s) of the protein adducin, a modulator of protein exposure on the cell surface that stimulates the sodium pump; a second is high concentrations of endogenous ouabain, an activating ligand for the pump. Both factors abnormally enhance the pump function through the triggering of the Src signaling pathway. Rostafuroxin, a derivative of digitoxigenin, acts as an antihypertensive agent by interfering with both of these ways to activate the sodium pump, preventing an increase in renal tubular Na+ transport and the resulting hypertension. In the first of the companion papers (Ferrandi et al.), the authors explore how rostafuroxin accomplishes its pressure-lowering feat. They show that the drug inhibits the Na+, K+ ATPase-Src-EGFR-ERK signaling activated by mutant adducin or ouabain, normalizing renal cell sodium transport, in two different rodent models of hypertension and in human cells. Upon closer examination of rostafurotoxin’s effects on Src-related phosphorylation in vitro, it became clear that the drug disrupts the ability of the variant adducin and the oubain-bound sodium pump to bind and activate Src at its SH2 domain. In the second of the companion papers (Lanzani et al.), the authors apply these results to patients by examining genetic variants that control the mechanisms of hypertension explored in the first paper. Lanzani et al. inspected genetic alterations in genes that encode enzymes that control ouabain synthesis and transport as well as two variants of adducin. They then tested the ability of these genetic variants to predict the response to rostafuroxin in a group of never-before treated patients with hypertension. Individuals who carried certain combinations of these genetic variants responded well to rostofuroxin, displaying a mean drop in the placebo-corrected blood pressure of about 14 mmHg, a clinically meaningful value. The same genetic signature did not predict the blood pressure response to other antihypertensive drugs with different mechanisms of action. The authors suggest that this genetic signature may exist in about a quarter of hypertensive patients. Finally, rostfuroxin may do more than lower blood pressure. Organ damage is known to be a downstream effect of an overactive Src signaling pathway—one of the byproducts of the hypertension mechanisms studied in this pair of papers. Because rostafuroxin interferes with Src signaling, the drug may curb the secondary damage to the heart, kidney, and brain caused by high blood pressure. Thus the kidney’s seemingly schizophrenic filtering actually represents a multilevel, fine-tuned control of the sodium pump as a means of managing blood pressure. Rostafuroxin can selectively correct hypertension in patients whose pumping mechanism is out of kilter, an advance toward personalized treatment of high blood pressure. Twenty years of genetic studies have not contributed to improvement in the clinical management of primary arterial hypertension. Genetic heterogeneity, epistatic-environmental-biological interactions, and the pathophysiological complexity of hypertension have hampered the clinical application of genetic findings. In the companion article, we furnished data from rodents and human cells demonstrating two hypertension-triggering mechanisms—variants of adducin and elevated concentrations of endogenous ouabain (within a particular range)—and their selective inhibition by the drug rostafuroxin. Here, we have investigated the relationship between variants of genes encoding enzymes for ouabain synthesis [LSS (lanosterol synthase) and HSD3B1 (hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 1)], ouabain transport {MDR1/ABCB1 [ATP-binding cassette, sub-family B (MDR/TAP), member 1]}, and adducin activity [ADD1 (adducin 1) and ADD3], and the responses to antihypertensive medications. We determined the presence of these variants in newly recruited, never-treated patients. The genetic profile defined by these variants predicted the antihypertensive effect of rostafuroxin (a mean placebo-corrected systolic blood pressure fall of 14 millimeters of mercury) but not that of losartan or hydrochlorothiazide. The magnitude of the rostafuroxin antihypertensive effect was twice that of antihypertensive drugs recently tested in phase 2 clinical trials. One-quarter of patients with primary hypertension display these variants of adducin or concentrations of endogenous ouabain and would be expected to respond to therapy with rostafuroxin. Because the mechanisms that are inhibited by rostafuroxin also underlie hypertension-related organ damage, this drug may also reduce the cardiovascular risk in these patients beyond that expected by the reduction in systolic blood pressure alone.

Genome Sequencing Reveals Loci under Artificial Selection that Underlie Disease Phenotypes in the Laboratory Rat

Summary Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models. PaperClip

Genomic Association Analysis of Common Variants Influencing Antihypertensive Response to Hydrochlorothiazide

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<10–5 were tested for replication of the associations in independent samples of hydrochlorothiazide-treated European hypertensives. The rs16960228 polymorphism in protein kinase C, &agr; 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.

Role of the adducin family genes in human essential hypertension

Objective In both humans and rats, polymorphisms of the alpha adducin (ADD1) gene are involved in renal sodium handling, essential hypertension and some of its organ complications. Adducin functions within cells as a heterodimer composed of various combinations of three subunits that are coded by three genes (ADD1, 2, 3) each located on a different chromosome. Design These characteristics provide the biochemical basis for investigating epistatic interactions among these loci. Methods We examined the three adducin gene polymorphisms and their association with ambulatory blood pressure (ABPM) and with plasma levels of renin activity (PRA), endogenous ouabain (EO), in 512 newly discovered and never-treated hypertensive patients. Results Relative to carriers of the wild type (Gly/Gly) ADD1 gene, patients carrying the mutated Trp ADD1 allele had higher blood pressure (systolic blood pressure (SBP) 143.2 ± 1.0 versus 140.6 ± 0.6 mmHg P = 0.027 and diastolic blood pressure (DBP) 94.2 ± 0.77 versus 92.3 ± 0.5 mmHg, P = 0.03), lower PRA and EO, consistent with the hypothesis of the renal sodium retaining effect of the Trp allele. Polymorphisms in the ADD2 and ADD3 genes taken alone were not associated with these variables. However, the differences in SBP and DBP between the two ADD1 genotypes were greatest in carriers of the ADD3 G allele (around + 8 mmHg). The significance of the interaction between ADD1 and ADD3 ranged between P = 0.020 to P = 0.006 according to the genetic model applied. Conclusions The interaction of ADD1 and ADD3 gene variants in humans is statistically associated with variation in blood pressure, suggesting the presence of epistatic effects among these loci.

Genes involved in vasoconstriction and vasodilation system affect salt-sensitive hypertension.

The importance of excess salt intake in the pathogenesis of hypertension is widely recognized. Blood pressure is controlled primarily by salt and water balance because of the infinite gain property of the kidney to rapidly eliminate excess fluid and salt. Up to fifty percent of patients with essential hypertension are salt-sensitive, as manifested by a rise in blood pressure with salt loading. We conducted a two-stage genetic analysis in hypertensive patients very accurately phenotyped for their salt-sensitivity. All newly discovered never treated before, essential hypertensives underwent an acute salt load to monitor the simultaneous changes in blood pressure and renal sodium excretion. The first stage consisted in an association analysis of genotyping data derived from genome-wide array on 329 subjects. Principal Component Analysis demonstrated that this population was homogenous. Among the strongest results, we detected a cluster of SNPs located in the first introns of PRKG1 gene (rs7897633, p = 2.34E-05) associated with variation in diastolic blood pressure after acute salt load. We further focused on two genetic loci, SLC24A3 and SLC8A1 (plasma membrane sodium/calcium exchange proteins, NCKX3 and NCX1, respectively) with a functional relationship with the previous gene and associated to variations in systolic blood pressure (the imputed rs3790261, p = 4.55E-06; and rs434082, p = 4.7E-03). In stage 2, we characterized 159 more patients for the SNPs in PRKG1, SLC24A3 and SLC8A1. Combined analysis showed an epistatic interaction of SNPs in SLC24A3 and SLC8A1 on the pressure-natriuresis (p interaction = 1.55E-04, p model = 3.35E-05), supporting their pathophysiological link in cellular calcium homeostasis. In conclusions, these findings point to a clear association between body sodium-blood pressure relations and molecules modulating the contractile state of vascular cells through an increase in cytoplasmic calcium concentration.

Relationships among endogenous ouabain, α-adducin polymorphisms and renal sodium handling in primary hypertension

Objective The basolateral Na pump drives renotubular reabsorption. In cultured renal cells, mutant adducins, as well as sub-nanomolar ouabain concentrations, stimulate the Na–K pump. Methods To determine whether these factors interact and affect Na handling and blood pressure (BP) in vivo, we studied 155 untreated hypertensive patients subdivided on the basis of their plasma endogenous ouabain or α-adducin genotype (ADD1 Gly460Trp-rs4961). Results Under basal conditions, proximal tubular reabsorption and plasma Na were higher in patients with mutated Trp ADD1 or increased endogenous ouabain (P = 0.002 and 0.05, respectively). BPs were higher in the high plasma endogenous ouabain group (P = 0.001). Following volume loading, the increment in BP (7.73 vs. 4.81 mmHg) and the slopes of the relationship between BP and Na excretion were greater [0.017 ± 0.002 vs. 0.009 ± 0.003 mmHg/(μEq min)] in ADD1 Trp vs. ADD1 Gly carriers (P < 0.05). BP changes were similar, whereas the slopes of the relationship between BP and Na excretion were lower [0.016 ± 0.003 vs. 0.008 ± 0.002 mmHg/(μEq min)] in patients with low vs. high endogenous ouabain (P < 0.05). In patients with high endogenous ouabain, volume loading increased the BP in the ADD1 Trp group but not in the Gly group (P < 0.05). Thus, patients with ADD1 Trp alleles are sensitive to salt and tubular Na reabsorption remains elevated after volume expansion. Conclusion With saline loading, BP changes are similar in high and low endogenous ouabain patients, whereas tubular Na reabsorption increases in the high endogenous ouabain group. Saline loading unmasks differences in renal Na handling in patients with mutant adducin or high endogenous ouabain and exposes an interaction of endogenous ouabain and Trp alleles on BP.

Epistatic interaction between alpha- and gamma-adducin influences peripheral and central pulse pressures in white Europeans.

Background Adducin is a membrane skeleton protein consisting of α- and β- or α- and γ-subunits. Mutations in α- and β-adducin are associated with hypertension. In the European Project on Genes in Hypertension, we investigated whether polymorphisms in the genes encoding α-adducin (Gly460Trp), β-adducin (C1797T) and γ-adducin (A386G), alone or in combination, affected pulse pressure (PP), an index of vascular stiffness. Methods We measured peripheral and central PP by conventional sphygmomanometry and applanation tonometry, respectively. We randomly recruited 642 subjects (162 nuclear families and 70 unrelated individuals) from three European populations. In multivariate analyses, we used generalized estimating equations and the quantitative transmission disequilibrium test. Results Peripheral and central PP averaged 46.1 and 32.6 mmHg, respectively. Among carriers of the α-adducin Trp allele, peripheral and central PP were 5.8 and 4.7 mmHg higher in γ-adducin GG homozygotes than in their AA counterparts, due to an increase in systolic pressure. γ-Adducin GG homozygosity was associated with lower urinary Na+/K+ ratio among α-adducin Trp allele carriers and with higher urinary aldosterone excretion among α-adducin GlyGly homozygotes. Sensitivity analyses in founders and offspring separately, and tests based on the transmission of the γ-adducin G allele across families, confirmed the interaction between the α- and γ-adducin genes. Conclusions In α-adducin Trp allele carriers, peripheral and central PP increased with the γ-adducin G allele. This epistatic interaction is physiologically consistent with the heterodimeric structure of the protein and its influence on transmembranous sodium transport.

Main results of the Ouabain and Adducin for Specific Intervention on Sodium in Hypertension Trial (OASIS-HT): a randomized placebo-controlled phase-2 dose-finding study of rostafuroxin

BackgroundThe Ouabain and Adducin for Specific Intervention on Sodium in Hypertension (OASIS-HT) Trial was a phase-2 dose-finding study of rostafuroxin, a digitoxygenin derivative, which selectively antagonizes the effects of endogenous ouabain (EO) on Na+,K+-ATPase and mutated adducin. Rostafuroxin lowered blood pressure (BP) in some animal models and in humans.MethodsOASIS-HT consisted of 5 concurrently running double-blind cross-over studies. After 4 weeks without treatment, 435 patients with uncomplicated systolic hypertension (140-169 mm Hg) were randomized to rostafuroxin (0.05, 0.15, 0.5, 1.5 or 5.0 mg/d) or matching placebo, each treatment period lasting 5 weeks. The primary endpoint was the reduction in systolic office BP. Among the secondary endpoints were diastolic office BP, 24-h ambulatory BP, plasma EO concentration and renin activity, 24-h urinary sodium and aldosterone excretion, and safety. ANOVA considered treatment sequence (fixed effect), subjects nested within sequence (random), period (fixed), and treatment (fixed).ResultsAmong 410 analyzable patients (40.5% women; mean age, 48.4 years), the differences in the primary endpoint (rostafuroxin minus placebo) ranged from -0.18 mm Hg (P = 0.90) on 0.15 mg/d rostafuroxin to 2.72 mm Hg (P = 0.04) on 0.05 mg/d. In the 5 dosage arms combined, the treatment effects averaged 1.30 mm Hg (P = 0.03) for systolic office BP; 0.70 mm Hg (P = 0.08) for diastolic office BP; 0.36 mm Hg (P = 0.49) for 24-h systolic BP; and 0.05 mm Hg (P = 0.88) for 24-h diastolic BP. In the 2 treatment groups combined, systolic (-1.36 mm Hg) and diastolic (-0.97 mm Hg) office BPs decreased from week 5 to 10 (P for period effect ≤0.028), but carry-over effects were not significant (P ≥ 0.11). All other endpoints were not different on rostafuroxin and placebo. Minor side-effects occurred with similarly low frequency on rostafuroxin and placebo.ConclusionsIn 5 concurrently running double-blind cross-over studies rostafuroxin did not reduce BP at any dose.Trial RegistrationClinicalTrials (NCT): NCT00415038

Steroid Biosynthesis and Renal Excretion in Human Essential Hypertension: Association With Blood Pressure and Endogenous Ouabain

BACKGROUND Endogenous ouabain (EO) has been linked with long-term changes in sodium balance and cardiovascular structure and function. The biosynthesis of EO involves, cholesterol side-chain cleavage (CYP11A1), 3-beta-hydroxysteroid dehydrogenase (HSD3B) with sequential metabolism of pregnenolone and progesterone. Furthermore, the renal excretion of cardiac glycosides is mediated by the organic anion transporter (SLCO4C1) at the basolateral membrane and the P-glycoprotein (PGP) (encoded by MDR1) at the apical membrane of the nephron. METHODS Average 24-h ambulatory blood pressures were recorded in 729 untreated essential hypertensives. Aldosterone (Aldo), EO, urinary Na+, and K+ excretions were determined. Single-nucleotide polymorphism (SNP) and haplotype-based association study was performed with a total of 26 informative SNPs. RESULTS Plasma EO was significantly directly related to both day (r = 0.131, P < 0.01) and nighttime diastolic blood pressure (DBP) (r = 0.143, P < 0.01), and remained significantly related after correction for confounders (sex, body mass index, age). Genotype analysis for EO levels and daytime DBP gave significant results for CYP11A1 rs11638442 and MDR1 rs1045642 (T/C Ile1145) in which the minor allele tracked with higher EO levels (T/T 210.3 (147-272) vs. C/C 270.7 (193-366) pmol/l, P < 0.001). Association was found between HSD3B1 polymorphisms and/or haplotypes with blood pressure (systolic blood pressure (SBP) 140.3 (11.7) vs. 143.8 (11.2) mm Hg, P < 0.01) and plasma Aldo (P < 0.05). Haplotype-based analyses support the data of SNP analysis. CONCLUSIONS Among patients with essential hypertension, cholesterol side-chain cleavage and MDR1 loci are related to circulating EO and DBP, most likely by influencing EO synthesis and transmembrane transport, respectively. In contrast, variants in HSD3B1 are related with SBP probably via Aldo.