Nunzia Casamassima

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.

Preoperative endogenous ouabain predicts acute kidney injury in cardiac surgery patients.

Objectives:Acute kidney injury is a frequent complication of cardiac surgery and increases morbidity and mortality. As preoperative biomarkers predicting the development of acute kidney injury are not available, we have tested the hypothesis that preoperative plasma levels of endogenous ouabain may function as this type of biomarker. Rationale and Design:Endogenous ouabain is an adrenal stress hormone associated with adverse cardiovascular outcomes. Its involvement in acute kidney injury is unknown. With studies in patients and animal settings, including isolated podocytes, we tested the above mentioned hypothesis. Patients:Preoperative endogenous ouabain was measured in 407 patients admitted for elective cardiac surgery and in a validation population of 219 other patients. We also studied the effect of prolonged elevations of circulating exogenous ouabain on renal parameters in rats and the influence of ouabain on podocyte proteins both “in vivo” and “in vitro.” Main Results:In the first group of patients, acute kidney injury (2.8%, 8.3%, 20.3%, p < 0.001) and ICU stay (1.4±0.38, 1.7±0.41, 2.4±0.59 days, p = 0.014) increased with each incremental preoperative endogenous ouabain tertile. In a linear regression analysis, the circulating endogenous ouabain value before surgery was the strongest predictor of acute kidney injury. In the validation cohort, acute kidney injury (0%, 5.9%, 8.2%, p < 0.0001) and ICU stay (1.2±0.09, 1.4±0.23, 2.2±0.77 days, p = 0.003) increased with the preoperative endogenous ouabain tertile. Values for preoperative endogenous ouabain significantly improved (area under curve: 0.85) risk prediction over the clinical score alone as measured by integrate discrimination improvement and net reclassification improvement. Finally, in the rat model, elevated circulating ouabain reduced creatinine clearance (–18%, p < 0.05), increased urinary protein excretion (+ 54%, p < 0.05), and reduced expression of podocyte nephrin (–29%, p < 0.01). This last finding was replicated ex vivo by incubating podocyte primary cell cultures with low-dose ouabain. Conclusions:Preoperative plasma endogenous ouabain levels are powerful biomarkers of acute kidney injury and postoperative complications and may be a direct cause of podocyte damage.

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.

Endogenous ouabain in renal Na+ handling and related diseases

The Na(+) pump and its Endogenous modulator Ouabain (EO) can be considered as an ancestral enzymatic system, conserved among species ranging from Drosophila to humans, related to Na handling. In this review, we examine how EO is linked with vascular function in hypertension and if it impacts the pathogenesis of heart and renal failure. Moreover, the molecular mechanism of endogenous ouabain-linked hypertension involves the sodium pump/sodium-calcium exchanger duet. Biosynthesis of EO occurs in adrenal glands and is under the control of angiotensin II, ACTH and epinephrine. Elevated concentrations of EO and in the sub-nanomolar concentration range were found to stimulate proliferation and differentiation of cardiac and smooth muscle cells. They may have a primary role in the development of cardiac dysfunction and failure. Experimental data suggest that the Na/K-ATPase α(2)-catalytic subunit causes EO-induced vasoconstriction. Finally, maneuvers that promote Na depletion, as diuretic therapy or reduced Na intake, raise the EO levels. Taken together, these findings suggest a key role for EO in body Na homeostasis.

AB0176 Association between Polymorphisms in Beta-Adducin and Sodium/Calcium Exchanger 1 and SLE with and without Nephritis

Background Genes involved in cytoskeletal assembly and water/electrolyte balance have been previously linked to hypertension, but recent evidences suggest a role also in the development of inflammation and autoimmunity [1-2]. Systemic Lupus Erythematosus is a prototypic multi-organ autoimmune disease, characterized by polymorphic clinical features and a complex polygenic background. Renal disease in SLE occurs in 40-70% of lupus patients and is responsible of an high burden of morbidity and mortality. Despite substantial efforts, less is known about the role of specific genetic factors in conferring susceptibility to SLE and its complications, including lupus nephritis (LN) [3]. Objectives With these premises we sought if an association between known hypertension-related polymorphisms and development of SLE and LN existed. Methods 106 patients (96 females, 10 males) diagnosed with SLE (n=51) or LN (n=55), 23 patients with Sjoegrens Syndrome and 62 healthy controls were enrolled. Patients and controls were genotyped for polymorphisms in α-, β- and γ-adducin (ADD1,2,3 respectively), angiotensin converting enzyme, transient receptor potential cation channel-subfamily C, solute carrier family 8 (sodium-calcium exchanger) member 1 (SLC8A1), solute carrier family 24 (sodium/potassium/calcium exchanger) member 3, PKD2 calcium channel and PRKG1 kinase genes. Results Beta-adducin (ADD2) rs4984 CT heterozygosis was significantly more frequent in SLE patients than in LN patients (χ2 =6.154; p=0.013). No patient had the ADD2 rs4984 TT genotype in accordance with a low prevalence of the ADD2 C1797T homozygosity in the population. Seven patients with SLE (n=3) or LN (n=4), but none of the control groups carried the AA variant of sodium/calcium exchanger 1 (SLC8A1) rs11893826 SNP (χ2 =11.771; p=0.019). Conclusions Genes involved in the control of electrolyte/water balance and cytoskeletal plasticity could affect the pathogenic background of SLE/LN. Larger studies are required to confirm these data and attempt correlations between genetic data and specific clinical features. References Tintinger GR, et al., Drug Des Devel Ther, 2009 Kleinewietfeld M, et al., Nature, 2013 Ramos PS, et al., Semin Nephrol, 2010 Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3378

A new clinical multivariable model that predicts postoperative acute kidney injury: impact of endogenous ouabain

BACKGROUND Acute kidney injury (AKI) is an important complication of cardiac surgery. Recently, elevated levels of endogenous ouabain (EO), an adrenal stress hormone with haemodynamic and renal effects, have been associated with worse renal outcome after cardiac surgery. Our aim was to develop and evaluate a new risk model of AKI using simple preoperative clinical parameters and to investigate the utility of EO. METHODS The primary outcome was AKI according to Acute Kidney Injury Network stage II or III. We selected the Northern New England Cardiovascular Disease Study Group (NNECDSG) as a reference model. We built a new internal predictive risk model considering common clinical variables (CLIN-RISK), compared this model with the NNECDSG model and determined whether the addition of preoperative plasma EO improved prediction of AKI. RESULTS All models were tested on >800 patients admitted for elective cardiac surgery in our hospital. Seventy-nine patients developed AKI (9.9%). Preoperative EO levels were strongly associated with the incidence of AKI and clinical complication (total ICU stay and in-hospital mortality). The NNECDSG model was confirmed as a good predictor of AKI (AUC 0.74, comparable to the NNECDSG reference population). Our CLIN-RISK model had improved predictive power for AKI (AUC 0.79, CI 95% 0.73-0.84). Furthermore, addition of preoperative EO levels to both clinical models improved AUC to 0.79 and to 0.83, respectively (ΔAUC +0.05 and +0.04, respectively, P < 0.01). CONCLUSION In a population where the predictive power of the NNECDSG model was confirmed, CLIN-RISK was more powerful. Both clinical models were further improved by the addition of preoperative plasma EO levels. These new models provide improved predictability of the relative risk for the development of AKI following cardiac surgery and suggest that EO is a marker for renal vascular injury.

Quantitative proteomics reveals novel therapeutic and diagnostic markers in hypertension.

Hypertension is a prevalent disorder in the world representing one of the major risk factors for heart attack and stroke. These risks are increased in salt sensitive individuals. Hypertension and salt sensitivity are complex phenotypes whose pathophysiology remains poorly understood and, remarkably, salt sensitivity is still laborious to diagnose. Here we present a urinary proteomic study specifically designed to identify urinary proteins relevant for the pathogenesis of hypertension and salt sensitivity. Despite previous studies that underlined the association of UMOD gene variants with hypertension, this work provides novel evidence showing different uromodulin protein level in the urine of hypertensive patients compared to healthy individuals. Notably, we also show that patients with higher level of uromodulin are homozygous for UMOD risk variant and display a decreased level of salt excretion, highlighting the essential role of UMOD in the regulation of salt reabsorption in hypertension. Additionally, we found that urinary nephrin 1, a marker of glomerular slit diaphragm, may predict a salt sensitive phenotype and positively correlate with increased albuminuria associated with this type of hypertension.

Arterial properties in relation to genetic variation in α-adducin and the renin–angiotensin system in a White population

Earlier studies have demonstrated the interaction between ADD1 and ACE in relation to arterial properties. We investigated whether arterial characteristics might also be related to interactions of ADD1 with other renin–angiotensin system genes. Using a family-based sampling frame, we randomly recruited 1064 Flemish subjects (mean age, 43.6 years; 50.4% women). By means of a wall-tracking ultrasound system, we measured the properties of the carotid, femoral and brachial arteries. In multivariate-adjusted analyses, we assessed the multiple gene effects of ADD1 (Gly460Trp), AGT (C–532T and G–6A) and AT1R (A1166C). In ADD1 Trp allele carriers, but not in ADD1 GlyGly homozygotes (P-value for interaction ⩽0.014), femoral cross-sectional compliance was significantly higher (0.74 vs 0.65 mm2 kPa−1; P=0.020) in carriers of the AT1R C allele than in AT1R AA homozygotes, with a similar trend for femoral distensibility (11.3 vs 10.2 × 10−3 kPa−1; P=0.055). These associations were independent of potential confounding factors, including age. Family-based analyses confirmed these results. Brachial diameter (4.35 vs 4.18 mm) and plasma renin activity (PRA) (0.23 vs 0.14 ng ml−1 h−1) were increased (P⩽0.005) in AGT CG haplotype homozygotes compared with non-carriers, whereas the opposite was true for brachial distensibility (12.4 vs 14.4 × 10−3 kPa−1; P=0.011). There was no interaction between AGT and any other gene in relation to the measured phenotypes. ADD1 and AT1R interactively determine the elastic properties of the femoral artery. There is a single-gene effect of the AGT promoter haplotypes on brachial properties and PRA.

Effects of genetic variation in adducin on left ventricular diastolic function as assessed by tissue Doppler imaging in a Flemish population

Background We investigated the possible association between left ventricular diastolic function and the ADD1 Gly460Trp and ADD3 IVS11 +386A>G polymorphisms alone and in combination. Methods In a family-based population study (473 subjects; 50.5% women; mean age 50.5 years), we measured early (Ea) and late (Aa) diastolic peak velocities of the mitral annulus by tissue Doppler imaging. In multivariate-adjusted analyses, we investigated phenotype–genotype associations, while accounting for confounders and family structure. Results Lateral Ea/Aa ratio was higher in ADD1 Trp allele carriers than in GlyGly homozygotes (1.51 vs. 1.40; P = 0.005) and was lower in ADD3 A allele carriers than in GG homozygotes (1.42 vs. 1.55; P = 0.005). The effects of ADD1 on the lateral Ea and Ea/Aa weakened with older age (P < 0.05). The best fitting model for lateral Ea and Ea/Aa included ADD1, ADD3, and the three-way interaction term of both genes with age. Below the age of 50 years, the lateral Ea/Aa ratio was higher in ADD1 Trp allele carriers than in GlyGly homozygotes (1.91 vs. 1.73; P = 0.006), particularly in the presence of ADD3 GG homozygosity (2.46 vs. 1.80; P = 0.0008). In older subjects, these phenotype–genotype associations were not significant (P > 0.20). Transmission of the ADD1 Trp allele to offspring was associated with higher lateral Ea (+0.91; P = 0.026) and Ea/Aa ratio (+0.23; P = 0.0008). Conclusion Our population-based study demonstrated that left ventricular diastolic relaxation is modulated by genetic variation in ADD1 and ADD3. This association was more prominent in younger subjects in whom longstanding environmental factors and ageing are less likely to mask genetic effects.