Circulating Ouabain Modulates Expression of Claudins in Rat Intestine and Cerebral Blood Vessels.

The ability of exogenous low ouabain concentrations to affect claudin expression and therefore epithelial barrier properties was demonstrated previously in cultured cell studies. We hypothesized that chronic elevation of circulating ouabain in vivo can affect the expression of claudins and tight junction permeability in different tissues. We tested this hypothesis in rats intraperitoneally injected with ouabain (1 µg/kg) for 4 days. Rat jejunum, colon and brain frontal lobes, which are variable in the expressed claudins and tight junction permeability, were examined. Moreover, the porcine jejunum cell line IPEC-J2 was studied. In IPEC-J2-cells, ouabain (10 nM, 19 days of incubation) stimulated epithelial barrier formation, increased transepithelial resistance and the level of cSrc-kinase activation by phosphorylation, accompanied with an increased expression of claudin-1, -5 and down-regulation of claudin-12; the expression of claudin-3, -4, -8 and tricellulin was not changed. In the jejunum, chronic ouabain increased the expression of claudin-1, -3 and -5 without an effect on claudin-2 and -4 expression. In the colon, only down-regulation of claudin-3 was observed. Chronic ouabain protected the intestine transepithelial resistance against functional injury induced by lipopolysaccharide treatment or by modeled acute microgravity; this regulation was most pronounced in the jejunum. Claudin-1 was also up-regulated in cerebral blood vessels. This was associated with reduction of claudin-3 expression while the expression of claudin-5 and occludin was not affected. Altogether, our results confirm that circulating ouabain can functionally and tissue-specifically affect barrier properties of epithelial and endothelial tissues via Na,K-ATPase-mediated modulation of claudins expression.

Skeletal Muscle Na,K-ATPase as a Target for Circulating Ouabain.

While the role of circulating ouabain-like compounds in the cardiovascular and central nervous systems, kidney and other tissues in health and disease is well documented, little is known about its effects in skeletal muscle. In this study, rats were intraperitoneally injected with ouabain (0.1-10 µg/kg for 4 days) alone or with subsequent injections of lipopolysaccharide (1 mg/kg). Some rats were also subjected to disuse for 6 h by hindlimb suspension. In the diaphragm muscle, chronic ouabain (1 µg/kg) hyperpolarized resting potential of extrajunctional membrane due to specific increase in electrogenic transport activity of the 2 Na,K-ATPase isozyme and without changes in 1 and 2 Na,K-ATPase protein content. Ouabain (10-20 nM), acutely applied to isolated intact diaphragm muscle from not injected rats, hyperpolarized the membrane to a similar extent. Chronic ouabain administration prevented lipopolysaccharide-induced (diaphragm muscle) or disuse-induced (soleus muscle) depolarization of the extrajunctional membrane. No stimulation of the 1 Na,K-ATPase activity in human red blood cells, purified lamb kidney and Torpedo membrane preparations by low ouabain concentrations was observed. Our results suggest that skeletal muscle electrogenesis is subjected to regulation by circulating ouabain via the 2 Na,K-ATPase isozyme that could be important for adaptation of this tissue to functional impairment.

Sodium pumps, ouabain and aldosterone in the brain: A neuromodulatory pathway underlying salt-sensitive hypertension and heart failure.

Accumulating evidence obtained over the last three decades has revealed a neuroendocrine system in the brain that mediates long term increases in blood pressure. The system involves distinct ion transport pathways including the alpha-2 isoform of the Na,K pump and epithelial sodium channels, as well as critical hormone elements such as angiotensin II, aldosterone, mineralocorticoid receptors and endogenous ouabain. Activation of this system either by circulating or central sodium ions and/or angiotensin II leads to a cascading sequence of events that begins in the hypothalamus and involves the participation of several brain nuclei including the subfornical organ, supraoptic and paraventricular nuclei and the rostral ventral medulla. Key events include heightened aldosterone synthesis and mineralocorticoid receptor activation, upregulation of epithelial sodium channels, augmented synthesis and secretion of endogenous ouabain from hypothalamic magnocellular neurons, and sustained increases in sympathetic outflow. The latter step depends upon increased production of angiotensin II and the primary amplification of angiotensin II type I receptor signaling from the paraventricular nucleus to the rostral ventral lateral medulla. The transmission of sympathetic traffic is secondarily amplified in the periphery by increased short- and long-term potentiation in sympathetic ganglia and by sustained actions of endogenous ouabain in the vascular wall that augment expression of sodium calcium exchange, increase cytosolic Ca2+ and heighten myogenic tone and contractility. Upregulation of this multi-amplifier system participates in forms of hypertension where salt, angiotensin and/or aldosterone are elevated and contributes to adverse outcomes in heart failure.

Reply: "Comment on: Endogenous Ouabain and Related Genes in the Translation from Hypertension to Renal Diseases".

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Does the hormone "endogenous ouabain" exist in the human circulation?

Studies in the early 1990s suggested that a hormone identical to ouabain or an isomer of ouabain is secreted by the adrenal glands into the circulation and plays a role in the regulation of arterial pressure and cardiac and renal function. This hormone, known as endogenous ouabain (EO), was claimed to contribute to the pathophysiology of a number of disorders including heart failure, renal failure, pregnancy-induced, and essential hypertension. However, some research groups have been unable to confirm the presence of EO in the human circulation and the issue remains in dispute. In that the implications are of considerable importance to clinicians who, like the authors, lack biochemical expertise, it would be useful if the dispute could be addressed by disinterested scientists with long-standing and acknowledged expertise in analytical chemistry who could opine as to whether the evidence is, or is not, sufficient to state categorically that EO does (or does not) exist in the circulation in man. This brief review does not present new data but, rather, recommends that adjudication is needed regarding this important issue. © 2018 BioFactors, 44(3):219-221, 2018.

Predicting acute kidney injury: current status and future challenges.

Acute kidney injury (AKI) is characterized by an acute decline in renal function and is associated to increased mortality rate, hospitalization time, and total health-related costs. The severity of this 'fearsome' clinical complication might depend on, or even be worsened by, the late detection of AKI, when the diagnosis is based on the elevation of serum creatinine (SCr). For these reasons, in recent years a great number of new tools, biomarkers and predictive models have been proposed to clinicians in order to improve diagnosis and prevent the development of AKI. The purpose of this narrative paper is to review the current state of the art in prediction and early detection of AKI and outline future challenges.

Sodium and potassium in the pathogenesis of hypertension: focus on the brain.

PURPOSE OF REVIEW: Primary hypertension is characterized by Na excess and K deficit in the body, which together are key to its pathogenesis. These derangements work jointly in the brain and the peripheral vascular wall to establish hypertension. In this review, we highlight recent evidence describing the central mechanisms through which Na surfeit and K deficit enhance sympathetic nerve activity, thereby raising peripheral vascular resistance and generating hypertension. RECENT FINDINGS: Animal studies point to a small increase in plasma and cerebrospinal fluid (CSF) [Na], a small decrease in CSF [K], and increased levels of circulating angiotensin II, aldosterone, and endogenous ouabain as the central signals evoking hypertension. These signals are detected by circumventricular organ sensors in the forebrain, and are then relayed to hypothalamic nuclei, which project angiotensinergic effector pathways to brainstem nuclei and spinal preganglionic neurons, triggering increased sympathetic nerve activity and hypertension. These central processes depend on a noncirculating (brain) renin-angiotensin-aldosterone system, local production of endogenous ouabain, and increased oxidative stress. SUMMARY: Recent insights into the mechanisms mediating the central effects of Na excess and K deficit on raising sympathetic activity might pave the way for novel approaches to preventing and treating hypertensive disorders.

ASSOSIATION OF ENDOGENOUS CARDIOTONIC STEROIDS WITH SALT-SENSITIVITY OF BLOOD PRESSURE IN GEORGIAN POPULATION.

This investigation differentiates types of essential hypertension in a Georgian population as well as describes endogenous cardiotonic steroids in salt-sensitive and salt-resistant subjects. This case control study included 185 subjects: 94 cases with stage 1 essential hypertension (JNC7) naïve to antihypertensive treatment, and 91 controls. A salt-sensitivity test was used to dichotomize case and control groups into salt-sensitive and salt-resistant subgroups. Blood and urine samples were obtained to categorize participants as consuming high and low salt diets. Endogenous cardiotonic steroids, sodium and plasma-renin activity (PRA) were measured in both samples at the different sodium conditions. Determinants of circulating levels of endogenous sodium pump inhibitors were carried out using the ELISA and RIA methods; PRA was assessed by radioimmunoassay. Descriptive statistics were used to analyze the data. Differences in variables between sodium conditions were assessed using paired t-tests. Salt-sensitivity was found in 60.5% of the total population investigated, with a higher proportion in females. A statistically significant positive correlation was found between salt-sensitivity and age in females (r=0.262, p<0.01), and with 24-hour urine sodium concentration changes (r=0.334, p<0.01). A significant negative correlation was found between salt-sensitivity and PRA. At the high sodium condition, endogenous MBG and OU were high in salt-sensitive subjects compared to those who were salt-resistant. These compounds decreased with a low-salt diet in both salt-sensitive cases and controls but remained the same in salt-resistant individuals. The MBG and OU levels positively correlated with systolic blood pressure in salt-sensitive individuals but no variability was evident among salt-resistant subjects. Our results show that MBG and OU levels start to increase at the normotensive stage and sustained high concentrations can lead to elevated systolic blood pressure, a risk factor for arterial hypertension in salt-sensitive subjects.

Ouabain Contributes to Kidney Damage in a Rat Model of Renal Ischemia-Reperfusion Injury.

Warm renal ischemia performed during partial nephrectomy has been found to be associated with kidney disease. Since endogenous ouabain (EO) is a neuro-endocrine hormone involved in renal damage, we evaluated the role of EO in renal ischemia-reperfusion injury (IRI). We measured plasma and renal EO variations and markers of glomerular and tubular damage (nephrin, KIM-1, Kidney-Injury-Molecule-1, α1 Na-K ATPase) and the protective effect of the ouabain inhibitor, rostafuroxin. We studied five groups of rats: (1) normal; (2) infused for eight weeks with ouabain (30 µg/kg/day, OHR) or (3) saline; (4) ouabain; or (5) saline-infused rats orally treated with 100 µg/kg/day rostafuroxin for four weeks. In group 1, 2-3 h after IRI, EO increased in ischemic kidneys while decreased in plasma. Nephrin progressively decreased and KIM-1 mRNA increased starting from 24 h. Ouabain infusion (group 2) increased blood pressure (from 111.7 to 153.4 mmHg) and ouabain levels in plasma and kidneys. In OHR ischemic kidneys at 120 h from IRI, nephrin, and KIM-1 changes were greater than those detected in the controls infused with saline (group 3). All these changes were blunted by rostafuroxin treatment (groups 4 and 5). These findings support the role of EO in IRI and suggest that rostafuroxin pre-treatment of patients before partial nephrectomy with warm ischemia may reduce IRI, particularly in those with high EO.

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

OBJECTIVE: In the setting of normal sodium (Na) intake, many patients with hypertension have inappropriately elevated plasma aldosterone (Aldo) levels and may be at increased risk for tissue damage. Moreover, other adrenocortical steroids, including endogenous ouabain can stimulate tissue damage. As endogenous ouabain is often elevated in chronically Na-loaded states, is a vasoconstrictor, raises blood pressure (BP), and also promotes tissue fibrosis, we investigated the extent to which plasma Aldo and endogenous ouabain were coelevated among naïve hypertensive patients (NHP). We also investigated the impact of an acute salt load on these steroids, BP, and renal function. METHODS: NHP (590) were grouped in tertiles based on their baseline plasma Aldo (mean ±â€ŠSEM first 7.59 ±â€Š0.18, versus third 24.15 ±â€Š0.31 ng/dl). Baseline plasma renin activity (2.4 ±â€Š0.1 versus 1.2 ±â€Š0.1 ng/ml per h, P < 0.001), endogenous ouabain (268 ±â€Š14.9 pmol/l versus 239.0 ±â€Š13.6 pmol, P < 0.01) and DBP (91.9 ±â€Š0.76 versus 89.6 ±â€Š0.71 mmHg, P = 0.017) were higher in NHP in the third versus the first Aldo tertile, respectively. RESULTS: Acute Na loading showed that the BP of the third Aldo tertile NHP was especially salt-sensitive (slope of pressure-natriuresis relationship 0.015 ±â€Š0.002 versus 0.003 ±â€Š0.001 µEq/mmHg per min, P = 0.00024 after adjustment for sex, BMI, and age). Regression analyses showed that plasma Aldo and endogenous ouabain were linearly related (ß = 0.181, P = 0.0003). CONCLUSION: Among patients with essential hypertension, circulating endogenous ouabain and Aldo are typically coelevated and their BP is salt-sensitive. In conditions where Aldo is inappropriately elevated, both Aldo and endogenous ouabain may contribute to adverse cardiovascular and renal outcomes.

Reduction in maternal circulating ouabain impairs offspring growth and kidney development.

Ouabain, a steroid present in the circulation and in various tissues, was shown to affect the growth and viability of various cells in culture. To test for the possible influence of this steroid on growth and viability in vivo, we investigated the involvement of maternal circulating ouabain in the regulation of fetal growth and organ development. We show that intraperitoneal administration of anti-ouabain antibodies to pregnant mice resulted in a >80% decline in the circulating ouabain level. This reduction caused a significant decrease in offspring body weight, accompanied by enlargement of the offspring heart and inhibition of kidney and liver growth. Kidney growth inhibition was manifested by a decrease in the size and number of nephrons. After the reduction in maternal circulating ouabain, kidney expression of cyclin D1 was reduced and the expression of the α1 isoform of the Na(+), K(+)-ATPase was increased. In addition, the elevation of proliferation signals including ERK1/2, p-90RSK, Akt, PCNA, and Ki-67, and a reduction in apoptotic factors such as Bax, caspase-3, and TUNEL were detected. During human pregnancy, the circulating maternal ouabain level increased and the highest concentration of the steroid was found in the placenta. Furthermore, circulating ouabain levels in women with small-for-gestational age neonates were significantly lower than the levels in women with normal-for-gestational age newborns. These results support the notion that ouabain is a growth factor and suggest that a reduction in the concentration of this hormone during pregnancy may increase the risk of impaired growth and kidney development.

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 Ca(2+) homeostasis; this increases contractility and augments sympathetic effects. The new axis likely contributes to the chronic pressor effect of brain Ang II.

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.

No endogenous ouabain is detectable in human plasma by ultra-sensitive UPLC-MS/MS.

BACKGROUND: The presence of a binding site for cardiac glycosides, such as digitoxin and digoxin, in the sodium-potassium-ATPase, stimulated attempts to isolate endogenous cardiotonic steroids. Using immunoassays, clinical studies found the cardenolide ouabain to be secreted endogenously in response to exercise and untreated hypertension and to be correlated with severity of clinical conditions such as kidney failure and dilated cardiomyopathy. The assays used were not standardized and the mean concentrations of endogenous ouabain reported for healthy controls ranged from 60 to 530 pmol/l. None of these immunoassays is available any more. Therefore, the aim of this study was to develop a highly specific and reliable method for measurement of ouabain in human plasma based on isotope dilution liquid chromatography tandem-mass spectrometry (ID-LC-MS/MS). METHOD: An ultra-sensitive and specific ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed which applied solid phase extraction of plasma for sample preparation. RESULTS: The method was comprehensively validated and had a lower limit of quantification of 1.7 pmol/l. However, despite this very low detection limit ouabain was not observed in plasma samples from patients with and without heart failure. CONCLUSION: Our results suggest that immunoassays previously used to quantify assumed endogenous ouabain detected compounds which are not structurally identical with ouabain. Cross reactivity of structurally related compounds of endogenous origin may cause these discrepancies between immunological and mass spectrometric analyses. Conclusive characterization of assumed endogenous counterparts of digoxin in a biomarker discovery approach seems to require distinct analytical techniques.