Levels Of Endogenous Ouabain Research Articles

#1954 Effects of lanosterol synthase (LSS) gene on the development of acute kidney injury after cardiac surgery

Abstract Background and Aims Acute kidney injury (AKI) is a common post-cardiac surgery complication, significantly affecting morbidity and mortality. Recent studies reported that high plasma levels of endogenous ouabain (EO), a stress hormone secreted by the adrenal glands, are associated to worse kidney outcomes after cardiac surgery. Our group demonstrated that EO activity is affected by a missense variant (rs2254524, C -> A, Val642Leu) in Lanosterol Synthase (LSS) gene, an enzyme implicated in EO synthesis. The aim of this work is to analyze the relationship between this LSS polymorphism and the development of AKI after cardiac surgery. Method 1237 patients undergoing elective cardiac surgery were enrolled in the study. Preoperative biological samples were collected (Table 1). LSS genotypes were determined for each patient. The primary outcome was AKI development, according to KDIGO 2012 guidelines. Results 21.4% of patients developed AKI. Different preoperative clinical variables were analyzed, identifying five independent risk factors significantly correlated to AKI development in multivariate logistic regression analysis: age (p < 0.001), FE (p = 0.005), NYHA class (p < 0.001), reoperation (p = 0.002) and complex surgical intervention (p < 0.001), Table 2. No significant differences were observed between preoperative EO plasma levels and allelic variants of LSS gene and patients with the less common A allele were not associated to a more severe preoperative clinical presentation, expressed as EuroScore II value (AA 2.54 ± 3.86 vs AC 2.15 ± 2.52 vs CC 2.04 ± 2.73; p = ns). LSS minor variants turned out significantly associated with the incidence of AKI (AA = 29.3%; AC = 25.3%; CC = 17.2%; X2 p < 0.001—Fig. 1). This evidence remains significant after correction for covariates associated to AKI previously reported (Log regression p = 0.002; RR for AA variant: 2.09 (IC95% 1.15-3.78)) Conclusion Patients with at least one mutated allele of LSS variant have a higher probability of developing AKI after cardiac surgery. We think these results could be of interest to further understand cellular mechanisms underlying AKI development, especially in the cardiac surgery field.

#1463 Assessment of salt-sensitive hypertension in a novel Lanosterol Synthase knock-in mouse model

Abstract Background and Aims The diversity in blood pressure (BP) response to various salt intakes, known as Salt Sensitivity (SS), exhibits significant differences among individuals within the hypertensive subjects relative to the general population. Hypertension (HT) and SS are correlated to elevated levels of plasma Endogenous Ouabain (EO). We aimed at delineating the role of the missense variation rs2254524 (Val642Leu; C>A) within the Lanosterol Synthase gene (LSS), encoding for an enzyme involved in steroid biosynthesis. Individuals with the AA LSS variant, adherent to a low-salt diet, had a more significant BP reduction compared to those with CC genotype. Method Utilising CRISPR-Cas9, we established a knock-in mouse model carrying the analogous LssV643L/V643L. Male mice were subjected to a normal-salt diet (0.5% NaCl), high-salt diet (4% NaCl), or low-salt diet (<0.03% Na) and BP was monitored every two days using a tail-cuff system for 10 days. Blood and urine were collected separately in metabolic cages. Results Homozygous mice for Lss mutation demonstrated normal viability and healthy phenotype, undistinguishable from the WT. At baseline, the Lss AA mice were affected by a significant augmentation in kidney weight at 3 and 12 months of age, relative to WT. The Lss V643L does not influence EO levels or systolic BP (SBP), specifically at 3 and 12 months, but affects the SBP response to dietary salt. AA mice showed a high SBP in a high-salt diet against control diet at the same time intervals. Moreover, the Lss variant impacted the pressure-natriuresis relationship, with the mutated AA strain showing a less steep curve both in control diet versus the high-salt diet and in control diet versus the low-salt diet, displaying the SS phenotype of the AA mice. The 12-month-old mice in high-salt diet displayed cardiac hypertrophy with the AA genotype exhibiting an elevated prevalence of cardiac fibrosis. The level of Lss mRNA decreased in the adrenal glands in response to a high-salt diet. RNASeq analysis identified distinct patterns in several Slc genes, Cbr2 and Arhgap26 within the kidneys of these mice, in control or high-salt dietary conditions. Conclusion The established LssV643L/V643L mouse model effectively mirrors the SS-HT phenotype. Our findings highlight the significance of the Lss in modulating BP in response to salt stimuli suggesting a potential organ damage pathway requiring further exploration in older mice.

#4677 EARLY MARKERS OF POSTOPERATIVE ACUTE KIDNEY INJURY

Abstract Background and Aims Acute kidney injury (AKI) is a common clinical condition among patients who undergo cardiac surgery, significantly affecting morbidity and mortality[1]. To date, preoperative biomarkers and solid predictive models for AKI are not yet currently available in clinical practice. Recent studies reported endogenous ouabain (EO), a stress hormone secreted by the adrenal glands, as associated to worse kidney outcomes after cardiac surgery[2]. The aim was to validate the use of EO as biomarker of individual susceptibility for AKI after cardiac surgery and to create a new powerful score for postoperative AKI risk. Method 1174 patients undergoing elective cardiac surgery were enrolled in the study. Preoperative biological samples were collected and analysed. The primary outcome was AKI development, according to KDIGO 2012 guidelines. Results 21.6% of patients developed AKI (9% developed severe AKI, stage≥2). AKI confirmed strong correlation to postoperative death: all patients dead within 90 days for postsurgical complication developed AKI and each AKI stage was associated to a 10 times higher mortality risk (p < 0.001). Different preoperative clinical variables were analyzed, identifying five independent risk factors significantly correlated to AKI and severe AKI: age, FE, NYHA class, reoperation and complex surgical intervention (p < 0.001 for all of them). Preoperative EO levels turned out significantly associated to the incidence of AKI (p < 0.001) and clinical complications. Specifically, patients with higher EO levels had greater incidence of AKI (p < 0.001) and worse cardiac and kidney basal function (p = 0.005 and p = 0.003, respectively). Finally, a simple score was developed on the base of those preoperative clinical values significantly associated to AKI (AUC for severe AKI = 0.79, p <0,001). Adding preoperative EO to the model significantly improved the predictive capability (AUC for severe AKI = 0.82, p < 0.001; Δ-AUC +0.0229, p = 0.026). Conclusion We confirmed in a large population the role of preoperative plasma level of EO as an important early predictor for post-surgical AKI and we built a powerful clinical model for postoperative AKI risk, exclusively using few simple preoperative clinical factors and a single biohumoral marker.

#4720 SEPSIS, AKI AND MORTALITY. SEARCHING FOR NEW MARKERS: OUR EXPERIENCE WITH ENDOGENOUS OUABAIN

Abstract Background and Aims Sepsis and septic shock are quite common events in the hospital setting. The classification of these events has changed a lot over the years, to ensure better sensitivity in recognizing a situation that, if not managed properly and quickly, leads to the death of the patient. In our study we focused on the search for predictors of acute kidney injury and mortality during the evolution of this pathology, with particular attention to the role of the Endogenous Ouabain hormone. Method We enrolled 177 patients diagnosed with sepsis or septic shock, under an ordinary hospital stay. We standardized patients according to the latest classification criteria (Sepsis-III), we focused our attention specifically on SOFA score and the relationship with acute kidney injury and mortality, including its relationship with other laboratory parameters, especially lactates. In a subgroup of patients was also dosed the level of Endogenous Ouabain (EO - hormone produced by the adrenal glands but which has an important impact on renal hemodynamics), through a venous sampling in 3 different times (at the beginning of the septic state, 24 and 48 hours later). Subsequently the variation of EO and the correlation with laboratory parameters was investigated in this period, to assess its impact on the development of kidney damage and mortality. Results The 70.1% of septic patients in our study develop AKI and, of this percentage, 75% in a severe form (stage 2-3 KDIGO). Both the presence and severity of AKI are unable to predict mortality in our sample (p-value 0.13). The presence of an impaired hemodynamics status (expressed ad MAP < 70 mmHg) has a strong impact in the severe stages of AKI but not in the mild form (p-value 0.006; ExpB 1.81 vs p-value 0.105; ExpB 1.46). The other elements related to the development of AKI were found to be the high levels of white blood cells, respiratory distress and the SOFA value at the presentation, while we found no correlation with CRP and lactate levels. Lactate levels well correlate with SOFA (p-value <0.0001 and Pearson's index 0.407) and mortality, particularly when the initial values are > 2mg/dL (p < 0.0001; ExpB 5.31, IC95% [2.07-13.57]). In the subgroup of patients in which EO was dosed, there was a strong correlation of the basal levels of this hormone with mortality even after correction for lactate levels and SOFA (p-value 0.009) but not with the development of AKI. However, patients in AKI stage-3 show persistently higher levels of EO both at 24 and 48 h (p = 0.015). Conclusion The presence of high levels of lactates at the onset is the most important predictor of mortality, followed by EO levels at presentation and the SOFA value. Elevation of Endogenous Ouabain levels is indicative of an acute state of stress but is not a parameter influenced by the SOFA score, nor by the general condition of patient. It seems non directly related with the development of AKI, tissue hypoperfusion or activation of the inflammatory response but is associated with an inability to rapidly recover renal function. The EO could be very useful in predicting mortality in patients with sepsis and septic shock as it seems to be an earlier, more specific, and sensitive than the other laboratory parameters used so far.

PRELIMINARY DATA ON THE ENDOGENOUS OUABAIN IN IDIOPATHIC PULMONARY ARTERIAL HYPERTENSION PATIENTS

Objective: The aim is to detect the endogenous ouabain (EO) in idiopathic pulmonary arterial hypertension (IPAH) patients. Design and method: EO is a steroid hormone secreted by the adrenal glands associated with adverse cardiovascular outcomes. However, EO plays other roles as brain protection against traumatic injury and seems involved in the adaptive response to hypoxia. Recently, we detected, for the first time, EO in a healthy human group of acute hypoxia (elite apnea divers) and in diving animals (common bottlenose dolphins, phocids and otariids, and loggerhead sea turtles). This study complements the above as we considered a human model of chronic hypoxia. We collected blood samples from five Caucasian males and measured EO with a Scintillation Proximity Assay. Results: We found that IPAH patients had higher plasma concentrations levels of EO than control subjects. In addition, EO plasma concentrations were negatively correlated with the mean pulmonary arterial pressure and total pulmonary vascular resistance (p-values <0.05). A positive correlation trend was observed with the cardiac index, a marker of healthy cardiac output. The results could suggest that high concentrations of EO are predictive of a better adaptation of the right ventricular afterload. Conclusions: Although these are preliminary results, they match data coming from other studies on clinical conditions that frequently occur in comorbidity with IPAH. It can represent a helpful hint for future investigations involving other pathological conditions for possible therapeutic and diagnostic approaches.

Sleep deprivation is associated with increased circulating levels of endogenous ouabain: Potential role in bipolar disorder

Endogenously produced cardiac glycosides, like endogenous ouabain (EO), are putative hormones that have been implicated in the pathophysiology of bipolar disorder. Individuals with bipolar disorder appear to be unable to sufficiently upregulate production of EO in situations of increased need. This study was performed to determine the effect of sleep deprivation on the circulating levels of EO. Plasma EO concentrations were measured by ouabain-radioimmunoassay in heterozygote Na,K-ATPase a2 knockout (KO) mice, which have been used as an animal model of mania, and wildtype siblings at baseline and after sleep fragmentation utilizing the moving bar method. a2 KO animals had elevated endogenous ouabain concentrations compared to wild type controls (0.82 ± SD 0.22 nM vs 0.26 ± 0.02, P = 0.03). Sleep fragmentation increased ouabain concentrations in wild type mice (0.53 ± 0.08 nM sleep fragmentation vs 0.26 ± 0.02 nM baseline, P = 0.04), but not in a2 KO mice (0.60 ± 0.07 nM sleep fragmentation vs 0.82 ± 0.22 nM baseline, P > 0.05). These studies demonstrate that sleep disturbance can increase EO in control mice but animals that exhibit some manic behaviors are unable to increase EO production.

Effects of renal denervation on endogenous ouabain in spontaneously hypertensive rats.

To investigate the role of renal denervation (RDN) on endogenous ouabain (EO) secretion in spontaneously hypertensive rats (SHR). Sixteen 12-week-old male SHR were randomly separated into the renal denervation group (RDNX group) and sham operation group (sham group), and eight age-matched Wistar Kyoto rats (WKY) were served as control group. EO concentrations, the Na+- K+-ATPaseactivity, and the expression of Na+-K+-ATPase were assessed. EO levels in serum, kidneys and hypothalamus of sham group were higher than in RDNX group (p < 0.05). Renal Na+-K+-ATPase activity subjected to denervation surgery showed significantly reduction when compared with the sham groups (p < 0.05). A positive correlation existed between norepinephrine (NE) content and Na+-K+-ATPase activity in the kidney (r2 = 0.579). Renal Na+-K+-ATPase α1 subunit mRNA expression was down-regulated in the RDNX group compared with the sham group (P < 0.05), while renal Na+-K+-ATPase α1 subunit mRNA expression was no statistical significance between the groups (P = 0.63). Immunohistochemical analysis showed that there were significant differences in the renal expression of Na+-K+-ATPasebetween the three groups (P < 0.05). These experiments demonstrate that RDN exerted an anti-hypertensive effect with reduction of EO levels and Na+-K+-ATPase activity and Na+-K+-ATPase α1 subunit expression of kidney in SHR.

Endogenous Ouabain: A Possible Link between High Salt Intake and Male Infertility

Male Infertility is an increasing global health issue, and one of the main findings in the semen of infertile men is spermatogenesis and sperm motility deficiencies. Despite the advances in male fertility, sperm abnormalities are complex and caused by multiple endogenous and exogenous factors. The Na, K ATPase α4 isoform is testis-specific, and its catalytic function is essential to maintain sperm quality, motility, and capacity. Exogenous ouabain is known for its inhibitory actions on the Na, K-ATPase, and α4 has a high affinity for ouabain. This heightened sensitivity is the basis for numerous experiments showing that Inhibition of the α4 by ouabain leads to sperm deterioration. It is interesting to note that endogenous ouabain is a cardiotonic steroid hormone found in animals and humans during normal conditions and increases in response to high salt intake. Also, infertile men with deficient sperm motility had increased levels of endogenous ouabain in the semen. Therefore, this article emphasizes the intersectionality between the following factors: 1) high salt intake and decreased sperm motility, 2) high salt intake and increased EO levels, 3) sperm Na, K-ATPase α4 isoform high affinity for ouabain, 4) effects of high ouabain on reduced sperm quality and motility, and 5) increased EO levels in infertile men with decreased sperm motility. Based on these observations, we hypothesize that endogenous ouabain is a possible link between high salt intake and male infertility.

Endolymphatic hydrops and ionic transporters: genetic and biohumoral aspects.

Ménière's disease (MD) is an inner ear disorder, characterized by a burden of symptoms, probably arising from the interplay of genetic and environmental factors. In this brief review, we consider the role of ion channels and transporters in the pathophysiology of MD, focusing on genetic and biohumoral aspects. Pathophysiological mechanisms related to altered concentrations of ions in the endolymph include altered osmotic pressure leading to hydrops and/or immunomodulatory effects of K+ and Endogenous Ouabain (EO) concentrations in the inner ear. Aquaporins 1-5 (AQPs) have been found in the inner ear; AQP2 is the only isoform controlled by a hormone, namely, vasopressin (antidiuretic hormone, ADH). Genetic studies on AQPs have provided inconclusive results. Recently, two genetic polymorphisms have been associated with MD: rs3746951, a missense variant (Gly180Ser) in the Salt-Inducible Kinase-1 (SIK1) gene and rs487119, an intronic variant of gene SLC8A1 coding for a Na+,Ca++ exchanger (NCX-1). EO is a hormone released by the midbrain and adrenal glands. It controls the constitutive capacity of modulating Na+,K+-ATPase activity. Higher plasma levels of EO have been found in MD subjects compared to a control group.

Lanosterol Synthase Genetic Variants, Endogenous Ouabain, and Both Acute and Chronic Kidney Injury

Studies of humans and animals have suggested that endogenous ouabain (EO) and related genes are mediators of acute (AKI) and chronic kidney injury. We sought to examine the relationship among EO levels, genetic variants in lanosterol synthase (LSS; an enzyme that catalyzes synthesis of cholesterol, a precursor of EO), and both AKI and chronic kidney injury. 2 prospective observational cohort studies and a cross-sectional study of kidney tissue. (1) A prospective cohort study of patients undergoing cardiovascular surgery, (2) measurement of EO concentration in kidney tissue removed because of an adjacent tumor, and (3) a prospective cohort study of patients with newly diagnosed essential hypertension. Missense variant in LSS (A instead of C allele at rs2254524), which leads to a valine to leucine substitution at amino acid642. Development of postoperative AKI in the cardiovascular surgery cohort, EO concentration in kidney tissue, and estimated glomerular filtration rate (eGFR) reductions in the essential hypertension cohort. Logistic regression for analysis of postoperative AKI, analysis of variance for EO concentration in kidney tissue, and generalized linear models for changes in eGFR over time. AKI incidence following cardiovascular surgery was greater among those with the LSS rs2254524 AA genotype (30.7%) than in those with the CC genotype (17.4%; P=0.001). LSS rs2254524 AA kidneys had higher EO concentrations than CC kidneys (2.14±0.29 vs 1.25±0.08ng/g; P<0.001). In the longitudinal study of patients with essential hypertension (median follow-up, 4 years; range, 1-15 years), eGFR decline was greater among the LSS rs2254524 AA genotype group (-4.39±1.18mL/min/1.73m2 per year) than in the AC or CC genotype groups (-1.07±0.55 and-2.00±0.45mL/min/1.73m2 per year respectively; P= 0.03). These associations do not necessarily represent causal relationships; LSS rs2254524 variants may have effects on other steroid hormones. These findings support the potential value of LSS rs2254524 genotype-based risk stratification to identify patients at high risk for AKI before cardiovascular surgery, as well as predict accelerated eGFR in the setting of hypertension. These findings also suggest that LSS may in part drive EO-mediated kidney damage. EO may represent a new potential therapeutic target for the prevention of AKI and slowing of kidney damage in the setting of hypertension.

Human neuronal cell based assay: A new in vitro model for toxicity evaluation of ciguatoxin

Ciguatoxins (CTXs) are emerging marine neurotoxins representing the main cause of ciguatera fish poisoning, an intoxication syndrome which configures a health emergency and constitutes an evolving issue constantly changing due to new vectors and derivatives of CTXs, as well as their presence in new non-endemic areas.The study applied the neuroblastoma cell model of human origin (SH-SY5Y) to evaluate species-specific mechanistic information on CTX toxicity. Metabolic functionality, cell morphology, cytosolic Ca2+i responses, neuronal cell growth and proliferation were assessed after short- (4–24h) and long-term exposure (10days) to P-CTX-3C.In SH-SY5Y, P-CTX-3C displayed a powerful cytotoxicity requiring the presence of both Veratridine and Ouabain. SH-SY5Y were very sensitive to Ouabain: 10 and 0.25nM appeared the optimal concentrations, for short- and long-term toxicity studies, respectively, to be used in co-incubation with Veratridine (25μM), simulating the physiological and pathological endogenous Ouabain levels in humans.P-CTX-3C cytotoxic effect, on human neurons co-incubated with OV (Ouabain+Veratridine) mix, was expressed starting from 100pM after short- and 25pM after long-term exposure. Notably, P-CTX-3C alone at 25nM induced cytotoxicity after 24h and prolonged exposure.This human brain-derived cell line appears a suitable cell-based-model to evaluate cytotoxicity of CTX present in marine food contaminated at low toxic levels and to characterize the toxicological profile of other/new congeners.

The Na+/Ca2+ exchanger: A possible link between oxidative stress and endogenous ouabain in hypertension

Oxidative stress-induced hypertension involves a number of membrane transport proteins that play a critical role in maintaining the cytosolic homeostasis of Na + and Ca 2+ . These transport proteins include Na + /K + -ATPase, the Na + /H + exchanger, Na channels, and the Na + /Ca 2+ exchanger. The exact link between these membrane transporters is not exactly known but appears to involve their location in microdomains called PLasmERosomes, oxidative stress and circulating ouabain levels. Oxidative stress stimulates the generation of ouabain from the adrenal glands which elevates circulating endogenous ouabain levels. At concentrations above 1nM, ouabain inhibits the activity of the Na + /K + -ATPase. At 1nM or lower concentrations, the Na + /K + -ATPase acts as a transducer which induces a cascade of events that begins with phosphorylation of the epidermal growth factor receptor (EGFR) by Src and activation of Ras, Raf, mitogen activated protein kinase kinase (MEK), mitogen activated protein kinase (MAPK), extracellular signal-regulated kinase1/2 (ERK1/2), and p90 ribosomal S6 kinase (p90RSK). p90RSK activates the Na + /H + exchanger through phosphorylation. Ouabain-mediated inhibition and activation of the Na + /K + -ATPase and Na + /H + exchanger, respectively, leads to elevation of Na + in the PLasmERosome which stimulates the Na + /Ca 2+ exchanger to extrude Na + in exchange for Ca 2+ . This elevates Ca 2+ in the PLasmERosome which stimulates junctional ER to release Ca 2+ . Ca 2+ influx through the Na + /Ca 2+ exchanger and release from junctional ER leads to Ca 2+ overload which can lead to a number of pathologies including increased vascular tone and hypertension.

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.

PP.09.21

Objective: Several studies indicate inappropriately elevated aldosterone (Aldo) secretion in ∼30% of newly discovered hypertensive patients. In primary aldosteronism homeostatic feedback loops between sodium balance and Aldo levels are disturbed and cardiovascular/renal damage is assumed to be secondary to high Aldo. However, other adrenocortical steroids including endogenous ouabain (EO) may be important. EO is elevated in sodium-loaded states, is a vasoconstrictor, raises blood pressure (BP), and promotes cardiovascular/renal damage. Design and method: Here, we investigated the relationship between circulating Aldo and EO and BP changes following an acute salt load in naïve hypertensive patients (NHP). Aldo cross-reactivity was < 1% in the EO assay. Results: NHP (488) were enrolled and grouped in tertiles by circulating Aldo (mean 1st 87 ± 2.18, 2nd 164 ± 1.83, and 3rd 332.3 ± 10.3 ng/dL). Baseline systolic and diastolic BPs were higher in the 3rd versus the 1st Aldo tertile (149.2 ± 1.17 vs 145.6 ± 1.11 and 98.4 ± 0.78 vs 92.5 ± 0.74 mmHg, p < 0.001, respectively). Circulating EO was higher in the 3rd Aldo tertile (285.7 ± 14.9 pmol/L) versus the 1st (239.0 ± 13.6 pM) and 2nd (211.4 ± 12.1 pM) tertiles (p < 0.01). Following an acute Na load, NHE in the 3rd Aldo tertile showed heightened salt–sensitivity of the pressure-natriuresis relationship (slope 0.013 ± 0.002 vs 0.004 μEq/mmHg/min, p = 0.008 after adjustment for sex, BMI, and age). In all subgroups circulating Aldo and PRA are suppressed after Na load. Plasma EO is not affected by Na load. Plasma K+ (4.07 ± 0.028 vs 3.93 ± 0.07 mEq/L) was higher (p = 0.002) in the 3rd Aldo tertile. Importantly, a direct (β = 0.116, p < 0.001) linear relationship was found between circulating Aldo and EO following adjustment for baseline BP, age, sex and BMI. β Conclusions: Among NHP, a large portion of patients have co-elevated circulating levels of EO and Aldo, and their BP is highly salt-sensitive. We suggest that EO and its target, the vascular myocyte α2 isoform of the Na-K ATPase, may contribute significantly to adverse cardiovascular and renal events in high aldosterone states.

Chronic ouabain treatment induces Rho kinase activation.

Ouabain is an endogenous Na(+)/K(+)-ATPase inhibitor whose chronic administration induces hypertension. Endogenous ouabain levels increase in human essential hypertension. On the other hand, Rho/Rho kinase (ROCK) pathway has been implicated in various animal models of hypertension. In the current work, we evaluated the possible involvement of Rho kinase in ouabain-induced hypertension. Ouabain was administered daily (20 µg/kg, i.p.) to Wistar rats for 6 weeks. After the ouabain treatment, we evaluated the possible changes in vascular responses to KCl and phenylephrine alone and in the presence of Rho kinase inhibitor Y27632. We also determined the expressions of ROCKs, Rho A and phosphorylation of myosin binding subunit of myosin light chain phosphatase (pMYPT) and activation of Rho A. Agonist-induced contractions in the presence of Y27632 are significantly decreased and Y27632-induced relaxations in aortas precontracted with phenylephrine are significantly enhanced with the chronic treatment of ouabain. Although the expressions of ROCK I and ROCK II remained unchanged, pMYPT expression was significantly increased in ouabain-treated group. Moreover, Rho A expression and activation were decreased after treatment with ouabain. Although Rho kinase expression did not change in aortas, increased basal Rho kinase activation may contribute to the development of ouabain-induced hypertension. Our current data present the first evidence that Rho kinase is involved in the development of ouabain-induced hypertension in rats.

Ouabain and Endogenous Ouabain -Dr. Jekyll and Mr. Hyde of Cardiac Glycosides?

In current research reports several disease states are claimed to be associated with elevated levels of endogenous ouabain. These include hypertension, cardiac enlargement, cardiac and renal failure, and a variety of terminal disorders. It has been suggested that endogenous ouabain should be considered as a major contributor to increased blood pressure and overall cardiovascular risk. These hypotheses stand in stark contrast to decades of clinical experiences with ouabain in humans, which indicate that plant-derived ouabain is effective in treating heart failure. In patients with hypertension ouabain lowers blood pressure. These conclusive clinical experiences refute the hypothesis that ouabain in humans is a major contributor to increased blood pressure and overall cardiovascular risk. The pronounced dependence of the ouabain effects on the dose, the state of the autonomic nervous system and species-specific characteristics in pharmacokinetics may explain many of the conflicting research results that have been published on experiments with ouabain. Rodents have different sensitivities to cardiac glycosides than humans. Pharmacokinetics of cardiac glycosides in rodents are also different from humans. Therefore, caution is advised when extrapolating experimental results in rodents to humans. The mutually exclusive effects of ouabain and the endogenous inhibitor of the Na/K-ATPase observed in mammalian tissues do not support the hypothesis that this inhibitor is identical with ouabain, but favour the interpretation that De Wardener’s “third factor” is something different, which also reacts to ouabain antibodies. Mini-review Article Furstenwerth; BJMMR, 8(5): 477-484, 2015; Article no.BJMMR.2015.470 478

Neuroendocrine humoral and vascular components in the pressor pathway for brain angiotensin II: a new axis in long term blood pressure control.

Central nervous system (CNS) administration of angiotensin II (Ang II) raises blood pressure (BP). The rise in BP reflects increased sympathetic outflow and a slower neuromodulatory pressor mechanism mediated by CNS mineralocorticoid receptors (MR). We investigated the hypothesis that the sustained phase of hypertension is associated also with elevated circulating levels of endogenous ouabain (EO), and chronic stimulation of arterial calcium transport proteins including the sodium-calcium exchanger (NCX1), the type 6 canonical transient receptor potential protein (TRPC6), and the sarcoplasmic reticulum calcium ATPase (SERCA2). Wistar rats received a chronic intra-cerebroventricular infusion of vehicle (C) or Ang II (A, 2.5 ng/min, for 14 days) alone or combined with the MR blocker, eplerenone (A+E, 5 µg/day), or the aldosterone synthase inhibitor, FAD286 (A+F, 25 µg/day). Conscious mean BP increased (P<0.05) in A (123±4 mm Hg) vs all other groups. Blood, pituitary and adrenal samples were taken for EO radioimmunoassay (RIA), and aortas for NCX1, TRPC6 and SERCA2 immunoblotting. Central infusion of Ang II raised plasma EO (0.58±0.08 vs C 0.34±0.07 nM (P<0.05), but not in A + E and A + F groups as confirmed by off-line liquid chromatography (LC)-RIA and LC-multistage mass spectrometry. Two novel isomers of EO were elevated by Ang II; the second less polar isomer increased >50-fold in the A+F group. Central Ang II increased arterial expression of NCX1, TRPC6 and SERCA2 (2.6, 1.75 and 3.7-fold, respectively; P<0.01)) but not when co-infused with E or F. Adrenal and pituitary EO were unchanged. We conclude that brain Ang II activates a CNS-humoral axis involving plasma EO. The elevated EO reprograms peripheral ion transport pathways known to control arterial Na+ and Ca2+ homeostasis; this increases contractility and augments sympathetic effects. The new axis likely contributes to the chronic pressor effect of brain Ang II.

Why isn't endogenous ouabain more widely accepted?

A new idea is the most quickly acting antigen known to science . —Wilfred Trotter During the past few years, several distinguished senior colleagues who visited our department and inquired about the status of endogenous ouabain (EO) have asked, “How come I didn't know about these [published]

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

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. 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. 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). 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.

RETRACTED: SIK1 localizes with nephrin in glomerular podocytes and its polymorphism predicts kidney injury

Mutant α-adducin and endogenous ouabain levels exert a causal role in hypertension by affecting renal Na-K ATPase. In addition, mutant β-adducin is involved in glomerular damage through nephrin down-regulation. Recently, the salt-inducible kinase 1 (SIK1) has been shown to exert a permissive role on mutant α-adducin effects on renal Na-K ATPase activity involved in blood pressure (BP) regulation and a SIK1 rs3746951 polymorphism has been associated with changes in vascular Na-K ATPase activity and BP. Here, we addressed the role of SIK1 on nephrin and glomerular functional modifications induced by mutant β-adducin and ouabain, by using congenic substrains of the Milan rats expressing either mutant α- or β-adducin, alone or in combination, ouabain hypertensive rats (OHR) and hypertensive patients. SIK1 co-localized and co-immunoprecipitated with nephrin from glomerular podocytes and associated with caveolar nephrin signaling. In cultured podocytes, nephrin-gene silencing decreased SIK1 expression. In mutant β-adducin congenic rats and in OHR, the podocyte damage was associated with decreased nephrin and SIK1 expression. Conversely, when the effects of β-adducin on podocytes were blocked by the presence of mutant α-adducin, nephrin and SIK1 expressions were restored. Ouabain effects were also reproduced in cultured podocytes. In hypertensive patients, nephrinuria, but not albuminuria, was higher in carriers of mutant SIK1 rs3746951 than in wild-type, implying a more direct effect of SIK1 on glomerular damage. These results demonstrate that, through nephrin, SIK1 is involved in the glomerular effects of mutant adducin and ouabain and a direct effect of SIK1 is also likely to occur in humans.