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Biochim Biophys Acta. Author manuscript; available in PMC 2012 December 7.
Published in final edited form as:
PMCID: PMC3517663

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 α2-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.

Keywords: Salt, Na pump, Kidney, Hypertension, Na+ transports

1. Introduction

For several million years the evolutionary ancestors of humans ate a diet that contained 1 g salt/day [1]. This implies that present-day humans are genetically programmed to a salt intake of that amount. The deliberate addition of salt to food only began ~5000–10,000 years ago with the beginning of agriculture so that the present consumption of ~10 g/day on average is, in evolutionary terms, relatively recent. Hypertension is the major risk factor for cardiovascular disease. High dietary salt is implicated in hypertension; however, the sensitivity of blood pressure to salt among individuals is variable, and the mechanisms of salt-sensitive hypertension are speculative [2]. Others have presumed a causal interrelationship between salt intake, total body Na+, fluid balance and blood pressure [3]. According to this model, kidney controls total body Na+ and thereby extracellular volume homeostasis. Reduced renal Na+ excretion results in extracellular volume expansion and augmented blood flow. Traditionally, the Na+ cation is thought to be largely restricted to the extracellular compartment, while K+ is stored intracellularly. This balance is maintained by the activity of the Na/K-ATPase in the cell membrane. The Na/K-ATPase contains a binding site for cardiac glycosides, such as ouabain, digoxin, and digitoxin, that is highly conserved among species ranging from Drosophila to humans. Although advantage has been taken of this site to treat congestive heart failure with drugs such as digoxin, this site has only recently been shown to have a natural function in blood pressure control [4]. The Na/K-ATPase can be considered as an ancestral enzymatic system related to Na+ handling that has adapted its function during evolution. In this review, we examine how sodium and endogenous ouabain influence blood pressure and some related disorders. We examine the evidence for EO biosynthesis in humans, the role of EO in primary hypertension and in renal Na handling, and in Ménière’s Syndrome. Finally, the trophic effect of EO on cardiovascular function will be mentioned.

2. Biosynthesis and clearance of endogenous ouabain (EO)

Since the original report published in 1991, EO has been isolated and detected by a variety of independent laboratories on different continents. HPLC and immunoassay methods show that EO is present in bovine [5] and human adrenal glands [6], bovine hypothalamus [7,8], rat adrenomedullary cells [9] and biological fluids [1013]. Mass spectrometry, NMR studies and the cochromatography of EO with ouabain in all tested chromatography systems confirm the presence of EO beyond doubt and show that the mammalian compound is identical to plant ouabain [79,1417].

A list of experimental evidences suggests that the adrenal is the main source of EO in humans and rats:

  1. The adrenal is enriched in EO in many mammals, and the tissue content remains remarkably constant under different conditions. Moreover, EO has been isolated and identified from bovine adrenals and hypothalamus [7,16].
  2. Plasma EO levels declined in adrenalectomized rats but not in rats whose adrenal medulla was removed [18]. Crucially, with prolonged adrenalectomy and corticosterone replacement in rats, plasma EO levels fell to the threshold sensitivity of the radioimmunoassay used in the study. Thus, the adrenal cortex is required to maintain normal plasma EO levels, and extra-adrenal sources are not sufficient in this capacity.
  3. In primary aldosteronism EO levels in the mixed inferior vena cava blood were more than 5–10 fold higher than in normal controls. If one assumes that the mixed venous plasma level of EO in adrenal patients lays between 2 and 4 nmol/L [19], then an approximately three-fold step up was present in adrenal venous plasma.
  4. In another study on conscious afebrile dogs in which adrenal venous catheters were surgically placed, the EO content of the adrenal venous effluent was approximately 5–6 fold higher than that of arterial blood [20].
  5. Elevated plasma levels of EO were found in hypertensive patients with adrenocortical tumors [21]. Removal of the tumors was associated with a normalization of plasma EO levels and the remission of hypertension. The results were compatible with those in other patients with adrenocortical tumors [22] and may represent the first example of ouabain-secreting adrenocortical tumors causing hypertension.
  6. Cultured human and bovine adrenocortical cells secrete EO into the culture fluid [23]. The secretion is augmented by angiotensin II, ACTH and possibly by vasopressin as well as alpha-1 adrenoceptor agonists [22]. ACTH appears to be a key regulator of circulating EO in humans [24].

The molecular basis for the elevated plasma EO in Milan Hypertensive Strains [10,25] was probed in the hypothalamus and adrenal using bioinformatics and genomic techniques. Elevated transcripts for cholesterol side-chain cleavage (also known as cytochrome P450scc, CYP11A1) and β-hydroxysteroid dehydrogenase/δ5-4 isomerase genes (HSD3B) were detected in hypothalamus [26]. Other studies have suggested that, as with aldosterone (Aldo), the biosynthesis of cardiac glycosides by the adrenal gland likely involves cholesterol side-chain cleavage to form pregnenolone with further metabolism of progesterone [2729]. Recently, we observed marked increases in circulating EO in uremic patients [30]. These data imply that, in addition to secretion, renal clearance is one of the major determinants of plasma EO. The biosynthesis of EO involves cholesterol side-chain cleavage (CYP11A1) and 3β-hydroxysteroid dehydrogenase (HSD3B) with sequential metabolism of pregnenolone and progesterone. Furthermore, the renal excretion of cardiac glycosides is mediated, in part by the organic anion transporter (SLCO4C1) [31] at the basolateral membrane and in part by the P-glycoprotein (PGP, encoded by MDR1) [32] at the apical membrane of the nephron. A single-nucleotide polymorphisms (SNPs) and haplotype-based association study was performed with a total of 26 informative SNPs in a large cohort of hypertensive patients. Among patients with essential hypertension, CYP11A1 and MDR1 loci were found associated to circulating EO and Diastolic Blood Pressure (DBP), most likely by influencing EO synthesis and transmembrane transport, respectively [33].

3. Endogenous ouabain in hypertension and renal Na handling

Human kidneys are poised to conserve sodium and excrete potassium. Prehistoric humans, who consumed a sodium-poor and potassium-rich diet, were well served by this mechanism [34]. With such a diet, sodium excretion is negligible and potassium excretion is high, matching potassium intake. In the contemporary dietary environment where high intakes of salt are common, the kidney must meet the obligation to excrete excess salt. Accordingly, it is of considerable interest that the reabsorption of filtered sodium by the renal tubules is increased in primary hypertension [35]. The augmented reabsorption appears to be mediated by the stimulation of several sodium transporters located at the luminal membrane, as well as the sodium pump, which is localized to the basolateral membrane and provides the energy for such transport. The circulating levels of EO change in response to variation of Na balance.

In studies on the relationship between Na intake/Na excretion and EO in various human experimental conditions, we noted the following:

  1. In a study of the general population [36], EO behaved as a blood pressure modulating factor whose circulating levels were linked with variations in plasma potassium. We suggested that EO acted either by inhibiting the pressor effect of an excessive salt intake or by counteracting the depressor action of sodium depletion. Furthermore, in 190 subjects whose plasma ouabain was equal to or less than 140 pmol/l (median), each 50 mmol/day increment in urinary sodium excretion was associated with an increase in blood pressure averaging 2.2 mm Hg (95% CI, 0.7–3.6 mm Hg; P=0.004) systolic and 1.4 mm Hg (95% CI, 0.3–2.5 mm Hg; P=0.01) diastolic [36]. In contrast, in subjects whose plasma ouabain was higher than 140 pmol/l, the association between blood pressure and urinary sodium excretion was not statistically significant. One possible interpretation of our findings is that EO might play a central role in the homeostatic regulation of blood pressure in response to changes in sodium intake in normotensive subjects [36].
  2. The impact of long-term alterations in sodium balance on the circulating levels and renal clearance of EO in normal humans have been investigated [37]. Thirteen normal men consumed a normal diet, high-salt diet, and hydrochlorothiazide (HCTZ), each for 5-day periods to alter sodium balance. In 13 normotensive subjects response to the high-salt diet, plasma EO increased >13-fold reaching 5.8±2.2 nmol/L (P<0.05) on the 3rd day of the diet. During HCTZ, body weight decreased and PRA, ALDO, and EO (1.71± 0.77 nmol/L) rose, while urinary EO excretion remained within the normal range (1.44±0.31 nmol/day). The net result of the above-mentioned study is that high-salt diet and HCTZ raise plasma EO by stimulating EO secretion, and a J-shaped curve relates sodium balance with EO in healthy men (Fig. 1).
    Fig. 1
    The relationship between sodium balance and circulating EO is described by a J-shaped curve: acute and chronic diuretic treatment, furosemide and HCTZ, (Negative Na balance), raise EO; at steady state of body Na balance (Normal) the circulating EO returns ...
  3. In never-treated, newly discovered patients with essential hypertension, there is a bimodal distribution of plasma EO levels [38]. The lower mode was 207±74 pmol/l (not different from that of normotensive controls, 253±53 pmol/l). The higher mode, representing approximately 40–45% of the patients with essential hypertension, was 540±197 pmol/l. Compared with patients with low EO, those with high EO essential hypertension had lower heart rates but increased stroke index and ventricular mass as expected with an entity having functional cardiotonic steroid activity.
  4. In hypertensive patients and in contrast to long-held hypotheses, acute salt loading was not an immediate stimulus to plasma EO. Basal levels of plasma EO do not differ among patients with salt-sensitive or salt-resistant hypertension [39]. We studied 138 patients with essential hypertension who underwent an acute volume expansion/contraction maneuver (2 days) and 20 patients who entered a blind randomized crossover design involving chronically controlled sodium intake and depletion (170 to 70 mmol/day; 2 weeks each period). In both maneuvers, plasma levels of EO were higher during Na depletion (acute: 338.8±17.4 and 402.7±22.8 pmol/l for baseline and low sodium, respectively, P = 0.01; chronic: 320.4 ± 32.0 versus 481.0 ± 48.1 pmol/l, P=0.01). Again a J-shaped curve related sodium balance with EO (Fig. 1). Taken together, these evidences suggest that EO is involved in the adaptation of humans to sodium depletion and this argues against the hypothesis that EO is a natriuretic hormone at least under these conditions.
  5. Among patients with more advanced hypertension, circulating levels of EO were directly related to both BP and total peripheral resistance and inversely related to cardiac index [40].
  6. We reported that circulating EO in hypertensive patients is raised specifically by maneuvers that promote the loss of body sodium [14,41]. Acute expansion of body fluids with isotonic saline is not a stimulus to plasma EO. Moreover, in hypertensive patients [41] carrying the mutated alpha-adducin (ADD1) variant, there is increased Na reabsorption with consistent suppression of PRA, Aldo and EO. Finally, patients with high plasma EO levels or mutated alpha-adducin display an increased BP and proximal tubular reabsorption [40]. A positive correlation (r2=0.9, P<0.001) was observed between plasma EO measured by both LC–MS/MS and radioimmunoassay in 21 patients. The plasma Na concentration was positively correlated with baseline plasma EO. Increased tubular reabsorption of Na+ has been proposed as an independent determinant of hypertension [42]. Our findings suggest a possible involvement of EO in this mechanism. The overall tubular reabsorption, expressed as FENa (Fractional Excretion of Na), and proximal tubular reabsorption, expressed as FELi (Fractional Excretion of Li), decreased in hypertensives with high EO levels.

All together these findings suggest that EO has a double effect at renal level: normal circulating values (less than 250 pM) by stimulating the basolateral Na/K-ATP, induce a signaling cascade that lead to an increase of Na+ reabsorption, through the renal Na/Ca exchanger (NCX1). On the other hand, elevated plasma EO induce natriuresis. Indeed, recently we showed that hypertensive patients with elevated plasma EO (>323 pmol/l) showed increased FENa and increased Na tubular rejection fraction (P=0.007) after acute saline load [Manunta P. unpublised data]. Furthermore, the effect of renin angiotensin aldosterone system (RAAS) and EO have been investigated: RAAS reflects body sodium status and has primarily a compensatory role in the regulation of BP. Conversely, EO has a biphasic relationship with tubular reabsorption (favoring Na retention at low plasma levels and Na excretion at the higher levels) which is likely to affect total body Na and blood pressure. The rise in circulating EO is due either to the genetic or renal clearance background. It has been shown that among patients with essential hypertension, CYP11A1 and MDR1 loci are related to circulating EO and DBP, most likely by influencing EO synthesis and transmembrane transport, respectively [33].

4. Endogenous ouabain in a Na related disease

Ménière’s disease (MD) is an inner ear disorder characterized by recurrent rotational vertigo and fluctuating sensorineural hearing loss. The pathogenetic mechanism is commonly accepted to be a raised endolymphatic pressure (hydrops). The peculiar ionic composition of endolymph, high K+ and low Na+ concentrations [43], is essential to mechano-electric transduction by hairy cells [44]. Na/K-ATPase has been abundantly demonstrated in the inner ear [45,46]. High concentrations of ouabain inhibit Na/K-ATPase activity in guinea pig inner ear [47]. Perilymphatic perfusion of ouabain decreases the endocochlear potential and induces ultrastructural changes in the stria vascularis [48]. Moreover, round window application of ouabain has been found to produce degeneration of outer hair cells and spiral limbus fibrocytes in the cochlea in guinea pigs [49].

The plasma levels of EO in MD patients [50,51]according to ADD1 Gly460Trp polymorphism were not different compared to controls even if their genotypic frequencies differed significantly (P=0.0013, chi-square=5.29). As Ménière disease has a complex pathology, we suppose that ADD1 and plasma EO may act as independent factors in inner ear on the pump activity modulation. The small group of patients studied is not enough to detect the differences seen in larger group of hypertensives. This example of dysregulation of tissue sodium balance is in agreement with the observations done in hypertensive patients, where the ADD1 locus and plasma EO interact and are related with whole body sodium status.

5. Endogenous ouabain and cardiovascular structure and function

EO activates signal transduction via the Src–epidermal growth factor receptor (EGFr)–extracellular signal-regulated kinase (ERK) pathway, and thereby triggers growth and proliferation of renal tubular cells and cardiomyocytes in vitro and in vivo, even at sub-nanomolar concentrations [52]. We therefore investigated the association between left ventricular structure and function and plasma EO in different conditions:

  1. In young offspring of hypertensive individuals, plasma EO levels positively correlate with some indices of diastolic cardiac dysfunction that precede the development of hypertension and left ventricular hypertrophy (LVH) [53].
  2. Left ventricular structure and function and plasma EO have been studied in a general population [54]. We randomly recruited 536 individuals from a general population (50.7% women, mean age 53.1 years). Measurements included echo-cardiographic left ventricular structure and function, blood pressure, plasma EO, and the 24-h urinary excretion of sodium. The key finding was that systolic blood pressure and left ventricular wall thickness increased with plasma EO. The significant positive association of relative wall thickness with plasma EO reflected increased left ventricular wall thickness and a slightly decreased left ventricular internal end-diastolic diameter. Our population-based study suggested that EO may have a trophic effect on the myocardium, independently from BP and other covariables. Elevated EO might be a factor that contributes to an increased risk of LV remodeling and hypertrophy amongst patients with essential hypertension.
  3. Among patients with idiopathic dilated cardiomyopathy, high circulating levels of EO (>233 pmol/l) identify those individuals predisposed to progress more rapidly to heart failure [55]. Moreover, patients on digoxin therapy display higher levels of EO (396.4± 187 pmol/l) than patients without therapy (211.6 ±71 pmol/l), and this difference persists when all confounders (including the potential cross-reactivity with digoxin) are taken into account [25]. The results also imply that digoxin should not be given to patients with high EO perhaps because of the potential for overt toxicity.
  4. In patients with severe cardiomyopathy and end-stage renal disease (ESRD) in dialysis [19], plasma EO was independently associated with left ventricular mass and geometry. This association was independent of arterial pressure and other well-established determinants of left ventricular mass. In ESRD, left ventricular hypertrophy and high cardiovascular risk in general represent multifactorial problems. Blood pressure, anaemia [56], hypoalbuminaemia, dyslipidaemia, hyperparathyroidism [57] and high sympathetic activity [58] all contribute to increased cardiovascular risk and LVH in these patients. Our study, once again, confirms that systolic BP, haemoglobin and cholesterol impact upon LVH in ESRD. In addition, our novel finding that EO was associated with LVH and that it is a marker of essential hypertension is fully in keeping with the notion that chronic volume overload is a potent stimulus that upregulates the steady-state levels of EO. A reduced capacity of the ventricular wall to develop hyperthrophy [59] and cardiomyocyte apoptosis triggered by ouabain [60] remain as hypothetical possibilities for the explanation of the eccentric LVH–EO link in ESRD. We also evaluated some of our radioimmunoassay determinations with liquid chromatography–tandem mass spectrometry (LC–MS/MS) in these patients. The EO content of the samples by LC–MS/MS and radioimmunoassay were highly correlated (r2=0.94) in linear regression analysis.

6. Conclusion

The results summarized here emphasize the role of EO in normotensive and hypertensive subjects. First, the hypertensinogenic effects of EO are in keeping with the experimental evidence indicating that the α2-catalytic subunit causes hypertension and ACTH-induced high BP [61,62]. Furthermore, the mutation of the α2-Na+ pump ouabain-binding site abolishes the hypertensinogenic effect of ouabain and ACTH [63]. Recently, it has been shown that the dynamic myogenic constriction in response to circulating nanomolar ouabain in small arteries likely makes a major contribution to the increased vascular tone and rise blood pressure in ouabain hypertensive rats [64]. Accordingly, this vascular mechanism may be relevant in human primary hypertension where elevated plasma EO levels are correlated with blood pressure in 40–50% of patients [3841].

Second, in addition to its direct influence on ion transport and vascular tone, ouabain is a growth factor that activates signaling cascades via a Src kinase pathway linked to the Na,K-ATPase [65,66]. The EO effect on structural and hyperthrophic cardiac myocytes has been discussed [40,5255]. Third, the data presented demonstrate a concentration-dependent relationship of EO with Na metabolism: its effect on tubular reabsorption which is likely to directly affect total body Na and blood pressure [39]. Recently, data in knock-out mice [62,63] suggest that the ouabain-binding site of the Na/K-ATPase affects the natriuretic response to a salt load by responding to endogenous Na,K-ATPase ligands. However, maneuvers that promote Na depletion [37,39] (reduced Na intake, diuretic therapy) raise plasma EO (J curve, Fig. 1).

The experimental evidences are in keeping with our initial hypothesis that EO and the Na pump are ancestral systems related to Na handling and that their interaction evolved and was physiologically relevant under conditions where salt intake was low. The association of EO with essential hypertension and cardiac hypertrophy are likely to be recent phenomena that reflect the widespread introduction of salt into cooking and food preservation in the last few millennia.


The authors acknowledge the expert technical assistance of Cinzia Scotti.

Sources of funding

Supported in part by USPHS grants HL75584, HL04521 and HL0788705 (JH) and European Union grants: LSMH-CT-2006-037093 InGenious HyperCare and HEALTH-F4-2007-201550 HyperGenes (PM).


Conflict of interests

None of the authors has a con3ict of interest with regard to the data presented in this manuscript.


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