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.

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.

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

In his recent letter, Dr [...].

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.

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.

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.

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.

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.

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 regulates cell viability.

The endogenous cardiac steroid-like compounds, endogenous ouabain (EO) in particular, are present in the human circulation and are considered putative ligands of the inhibitory binding site of the plasma membrane Na(+)-K(+)-ATPase. A vast amount of data shows that, when added to cell cultures, these steroids promote the growth of cardiac, vascular, and epithelial cells. However, the involvement of the endogenous compounds in the regulation of cell viability and proliferation has never been addressed experimentally. In this study, we show that EO is present in mammalian sera and cerebral spinal fluid, as well as in commercial bovine and horse sera. The lowering of serum EO concentration by the addition of specific anti-ouabain antibodies caused a decrease in the viability of several cultured cell lines. Among these, neuronal NT2 cells were mostly affected, whereas no reduction in viability was seen in rat neuroendocrine PC12 and monkey kidney COS-7 cells. The anti-ouabain antibody-induced reduction in NT2 cell viability was significantly attenuated by the addition of ouabain and was not observed in cells growing in serum-free media. Furthermore, the addition to the medium of low concentrations (nM) of the cardenolide ouabain, but not of the bufadienolide bufalin, increased NT2 and PC12 cell viability and proliferation. In addition, at these concentrations both ouabain and bufalin caused the activation of ERK1/2 in the NT2 cells. The specific ERK1/2 inhibitor U0126 inhibited both the ouabain-induced activation of the enzyme and the increase in cell viability. Furthermore, anti-ouabain antibodies attenuated serum-stimulated ERK1/2 activity in NT2 but not in PC12 cells. Cumulatively, our results suggest that EO plays a significant role in the regulation of cell viability. In addition, our findings support the notion that activation of the ERK1/2 signaling pathway is obligatory but not sufficient for the induction of cell viability by EO.

How NaCl raises blood pressure: a new paradigm for the pathogenesis of salt-dependent hypertension.

Excess dietary salt is a major cause of hypertension. Nevertheless, the specific mechanisms by which salt increases arterial constriction and peripheral vascular resistance, and thereby raises blood pressure (BP), are poorly understood. Here we summarize recent evidence that defines specific molecular links between Na(+) and the elevated vascular resistance that directly produces high BP. In this new paradigm, high dietary salt raises cerebrospinal fluid [Na(+)]. This leads, via the Na(+)-sensing circumventricular organs of the brain, to increased sympathetic nerve activity (SNA), a major trigger of vasoconstriction. Plasma levels of endogenous ouabain (EO), the Na(+) pump ligand, also become elevated. Remarkably, high cerebrospinal fluid [Na(+)]-evoked, locally secreted (hypothalamic) EO participates in a pathway that mediates the sustained increase in SNA. This hypothalamic signaling chain includes aldosterone, epithelial Na(+) channels, EO, ouabain-sensitive α(2) Na(+) pumps, and angiotensin II (ANG II). The EO increases (e.g.) hypothalamic ANG-II type-1 receptor and NADPH oxidase and decreases neuronal nitric oxide synthase protein expression. The aldosterone-epithelial Na(+) channel-EO-α(2) Na(+) pump-ANG-II pathway modulates the activity of brain cardiovascular control centers that regulate the BP set point and induce sustained changes in SNA. In the periphery, the EO secreted by the adrenal cortex directly enhances vasoconstriction via an EO-α(2) Na(+) pump-Na(+)/Ca(2+) exchanger-Ca(2+) signaling pathway. Circulating EO also activates an EO-α(2) Na(+) pump-Src kinase signaling cascade. This increases the expression of the Na(+)/Ca(2+) exchanger-transient receptor potential cation channel Ca(2+) signaling pathway in arterial smooth muscle but decreases the expression of endothelial vasodilator mechanisms. Additionally, EO is a growth factor and may directly participate in the arterial structural remodeling and lumen narrowing that is frequently observed in established hypertension. These several central and peripheral mechanisms are coordinated, in part by EO, to effect and maintain the salt-induced elevation of BP.

Endogenous cardiotonic steroids in chronic renal failure.

BACKGROUND: Previous reports demonstrated that digitalis-like cardiotonic steroids (CTS) contribute to the pathogenesis of end-stage renal disease. The goal of the present study was to define the nature of CTS in patients with chronic kidney disease (CKD) and in partially nephrectomized (PNx) rats. METHODS: In patients with CKD and in healthy controls, we determined plasma levels of marinobufagenin (MBG) and endogenous ouabain (EO) and erythrocyte Na/K-ATPase activity in the absence and in the presence of 3E9 anti-MBG monoclonal antibody (mAb) and Digibind. Levels of MBG and EO were also determined in sham-operated Sprague-Dawley rats and in rats following 4 weeks of PNx. RESULTS: In 25 patients with CKD plasma, MBG but not EO was increased (0.86 ± 0.07 versus 0.28 ± 0.02 nmol/L, P < 0.01) and erythrocyte Na/K-ATPase was inhibited (1.24 ± 0.10 versus 2.80 ± 0.09 µmol Pi/mL/h, P < 0.01) as compared to that in 19 healthy subjects. Ex vivo, 3E9 mAb restored Na/K-ATPase in erythrocytes from patients with CKD but did not affect Na/K-ATPase from control subjects. Following chromatographic fractionation of uremic versus normal plasma, a competitive immunoassay based on anti-MBG mAb detected a 3-fold increase in the level of endogenous material having retention time similar to that seen with MBG. A similar pattern of CTS changes was observed in uremic rats. As compared to sham-operated animals, PNx rats exhibited 3-fold elevated levels of MBG but not that of EO. CONCLUSIONS: In chronic renal failure, elevated levels of a bufadienolide CTS, MBG, contribute to Na/K-ATPase inhibition and may represent a potential target for therapy.

Endogenous ouabain: a link between sodium intake and hypertension.

The sodium pump, an ancestral enzyme with conserved ability to bind ouabain, plays a key role in salt conservation and is regulated by aldosterone and endogenous ouabain (EO). Plasma EO is elevated in about 45% of patients with essential hypertension and correlates with blood pressure. The relationship of EO with Na(+) balance is complex. Na(+) depletion raises circulating EO, whereas acute saline loads have no effect on EO in essential hypertension, and ambient levels of EO are unrelated to the saline sensitivity of blood pressure. Short-term periods of high dietary salt elevate EO and the relationship with salt balance in normal individuals is V-shaped, whereas the long-term relationship is likely to be L-shaped. Normal individuals suppress the high EO transient triggered by high-salt diets and avoid hypertension. In contrast, patients with elevated EO on normal Na(+) intakes have hypertension related to poor modulation of EO biosynthesis, clearance, or both.

Effects of mono- and dual blockade of the renin-angiotensin system on markers of cardiovascular status in hypertensive patients with mild and moderate renal failure.

BACKGROUND/AIMS: Dual renin-angiotensin system (RAS) blockade has no more efficiency to decrease cardiovascular mortality than mono-blockade. Our goal was to explore differences between other cardiovascular markers in patients with RAS blockade. METHODS: We analyzed two groups of patients treated with a long-term ACE inhibitor (MONO-group, n = 20) and an ACE inhibitor and angiotensin II receptor blocker (DUAL-group, n = 15). Ambulatory blood pressure monitoring, echocardiography, arterial stiffness and levels of catecholamine, endogenous ouabain (EO), pro-brain natriuretic peptide and more types of urinary albumin measurements were performed. RESULTS: In the DUAL-group, we found significantly better cardiac parameters, but the levels of EO and urinary albumins were similar in both groups. The level of EO correlates with nighttime mean arterial blood pressure (R = 0.556, p = 0.032) and arterial ß-stiffness (R = 0.512, p = 0.042). Urinary immuno-unreactive albumin showed a relationship with diastolic dysfunction of the heart (R = -0.508, p = 0.045) diurnal index of diastolic blood pressure (R = -0.569, p = 0.021) in the MONO-group. CONCLUSION: Cardiac parameters were more prosperous in the DUAL-group, but the levels of EO did not differ between groups. The level of EO correlated with blood pressure and arterial stiffness markers in the MONO-group only. The urinary immuno-unreactive albumin may be a new marker of cardiovascular conditions.

[Mechanisms for linking high salt intake to vascular tone: role of Na(+) pump and Na(+)/Ca²(+) exchanger coupling].

Excessive salt intake is a major risk factor for hypertension. However, the underlying molecular relationship between salt and hypertension is not fully understood. Recently discovered cardiotonic steroids, such as endogenous ouabain and other steroids, have been proposed as candidate intermediaries. Plasma cardiotonic steroids are significantly elevated in patients with essential hypertension and in salt-dependent hypertensive animals. Generally, it is believed that cardiotonic steroids inhibit Na(+) pump activity and lead to an increase in the cytosolic Na(+) concentration. Cellular Na(+) accumulation raises the cytosolic Ca²(+) concentration through the involvement of Na(+)/Ca²(+) exchanger type 1 (NCX1). In isolated arteries from α2 Na(+) pump knockout mice (α2(+/-)), myogenic tone is increased, and NCX inhibitor normalizes the elevated myogenic tone in α2(+/-) arteries. The NCX inhibitor lowers arterial blood pressure in salt-dependent hypertensive rats but not in other types of hypertensive rats or in normotensive rats. Furthermore, smooth muscle-specific NCX1 transgenic mice are hypersensitive to salt, whereas mice with smooth muscle-specific knockout of NCX1 (NCX1(SM-/-)) have low salt sensitivity. These results suggest that functional coupling between the vascular α2 Na(+) pump and NCX1 is a critical molecular mechanism for salt-induced blood pressure elevation.

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.

Ouabain, a circulating hormone secreted by the adrenals, is pivotal in cardiovascular disease. Fact or fantasy?

Substantial evidence points to the presence in human plasma of an inhibitor of the sodium/potassium pump which plays a central role in the pathophysiology of circulatory disorders, including essential hypertension. Studies from the 1980/90s claimed that this inhibitor was identical or very similar in structure to plant-derived ouabain and was synthesized by the adrenal cortex. However, the physical evidence in studies reporting isolation and identification of ouabain in human plasma appears insecure on closer examination. Additionally, reported circulating levels of immunoreactive ouabain in humans vary greatly, the ability of the human adrenal glands to secrete ouabain is questionable and the original commercial assay for measuring immunoreactive ouabain is no longer available. We submit that the position of ouabain as an endogenous, adrenally produced regulator of the sodium pump is of such importance that the current evidence needs either to put on a more secure footing or to lose its current status.

Endogenous ouabain in cardiovascular function and disease.

An endogenous ouabain has been isolated and conclusively identified from several mammalian tissues, including human plasma, by a number of independent laboratories. Substantial evidence from independent laboratories in several continents is consistent with an adrenal source for most if not all of the circulating endogenous ouabain. Accumulating evidence suggests that circulating levels of endogenous ouabain in humans are modulated by dietary salt and chronic volume status. Endogenous ouabain is linked significantly with vascular function in hypertension and likely impacts the pathogenesis of heart and renal failure. Moreover, the molecular mechanism of endogenous ouabain-linked hypertension involves the sodium pump/sodium-calcium exchange duet. The outstanding analytical issues include the elucidation of distal events in the biosynthetic pathway for endogenous ouabain and identification of molecular mechanisms that regulate its secretion and clearance.

Endogenous ouabain and cardiomyopathy in dialysis patients.

BACKGROUND AND METHODS: Endogenous ouabain (EO) is markedly raised in patients with chronic renal failure. As high EO induces myocardial cell hypertrophy in vitro and it is associated with left ventricular hypertrophy (LVH) in essential hypertensives and in patients with heart failure we investigated the relationship between plasma EO and LV mass and geometry in 156 end-stage renal disease (ESRD) patients. EO was measured by a specific radioimmunoassay and by mass spectrometry. RESULTS: On univariate analysis, plasma EO was directly related to LV mass (r = 0.26, P = 0.001) and LV end diastolic volume (r = 0.25, P = 0.002) and these relationships held true in multiple linear regression models including a series of potential confounders. Patients with eccentric LVH (n = 41, i.e. 26%) had the highest plasma levels of EO when compared to patients with other patterns of LV geometry (P = 0.001). Furthermore, plasma EO had diagnostic value for eccentric LVH because the area under the corresponding ROC curve (68%) was significantly greater (P = 0.002) than the threshold of diagnostic indifference. In this analysis, the sensitivity was 91% and the specificity was 36%. The positive predictive value was 33% but EO had a remarkably high negative predictive value (92%) for the exclusion of eccentric hypertrophy. CONCLUSIONS: In ESRD patients, plasma EO is independently associated with LV mass, LV volume and eccentric LVH. The results of this study are compatible with the hypothesis that EO is involved in alterations of LV mass in ESRD.