**3. Endogenous Ouabain Pressor Mechanism and Genes Involved in the Pathogenesis of Hypertension**

During the past years, many research groups investigated the molecular basis of essential hypertension, focusing their attention on renal, endocrine, nervous and humoral dysfunction. In particular, they hypothesized that alterations in renal sodium management could have a key role in its pathogenesis. The Na+/K+-ATPase activity in the kidneys is regulated by hormonal and genetic factors including EO and the gene coding for α-adducin (*ADD1*). Adducin is a cytoskeletal protein consisting of two heterodimers (α/β or α/γ). *ADD1*, *ADD2* and *ADD3* are the three coding genes for these subunits. It was shown that a polymorphism in the gene *ADD1* (determining the presence of a tryptophan instead of a glycine in the amino-acid position 460, Gly460Trp) was associated with a higher

expression of Na+/K+-ATPase in the surface of the cell and an enhancement of its activity. To better comprehend the mechanisms undergoing primary hypertension, researchers developed several rat models of genetic hypertension, including the Milan hypertensive strain (MHS) of rats that represents a suitable model for a subgroup of human patients with hypertension. It was shown, in both MHS rats and humans, that increased concentrations of EO corresponded to an increased tubular sodium reabsorption and, consequently, hypertension [32]. Furthermore, a prolonged infusion of low doses of plant-derived ouabain in normotensive rats and rat renal tubular cultured cells was associated to an enhanced expression and activity of the Na+/K+ pump, leading to a reversible form of hypertension. Starting from these evidences, it was hypothesized that ouabain itself may be considered as a pressor agent in vivo. The same phenomenon was documented in cells transfected with genetic variants of the MHS adducin [33]. In the following years, researchers also tried to understand the mechanism, by which both ouabain and mutated adducin could modify the expression of the Na+/K+-ATPase in the kidneys. Under these two conditions, it was possible to evidence a slower recycling of the sodium pump from the surface of the cell and, consequently, an excessive expression of the Na+/K+-ATPase in light of a tighter anchoring to the cytoskeletal proteins. This is the biochemical alteration present in both the ouabain- and adducin-dependent forms of hypertension [34]. These findings apparently contradict the traditional natriuretic hypothesis that considers the cardiotonic steroids as inhibitors of the Na+/K+-ATPase. According to this hypothesis, volume expansion conditions might induce the release of an endogenous hormone (EH) able to promote natriuresis. High levels of EO should lead to a decrease, rather than an increase, of Na+/K+-ATPase activity. To clarify this issue, the relation between EO and changes in sodium balance was studied in both rats and patients with essential hypertension. The results showed that an acute and chronic restriction of salt intake (but not the acute salt loading) was associated with a significant rise in EO plasmatic levels [35]. Consequently, conditions of salt and water reductions might provoke the elevation of the EO humoral concentration, meaning that EO does not act as a natriuretic hormone in vivo [4,36,37]. An important augmented EO level during physical exercise was demonstrated, which is a state characterized by an increased sympathetic activity and a decline in renal blood flow [4,38], and is found in patients undergoing cardiac surgery [39]. These results evidenced that EO induces a variety of important mechanisms, which augment vascular tone promoting renal sodium retention [4]. It might be a fair assumption that EO, through the enhancement of the renal Na+/K+-ATPase activity, plays a role in body sodium homeostasis and in the re-establishment and maintenance of the hydro-saline equilibrium [3,19]. Previous studies demonstrated that adducin has a direct role in the modulation of the renal Na+/K+-ATPase (Figure 1). Following this evidence, it was important to understand whether adducin polymorphism (Gly460Trp) might influence EO's response of the adaptation to a low-salt diet.

In hypertensive patients with mutated *ADD1* gene (*Trp-460*), a chronic low-salt diet is associated to an important augment of EO plasmatic concentration. Contrarily, it is not possible to recognize the same condition in wild-type patients (Gly-460), thus counteracting the hypotensive effect of the low-salt diet. Similarly, there is an increase of EO levels in MHS rats and the congenic rat strain NA (obtained by the introgression of the MHS *ADD1* locus into the normotensive genetic background), but not in normotensive rats. A high-salt diet does not modify plasma EO in rats, nor in humans. These data suggest that adducin genotype might predict the changes of EO plasmatic levels under salt restriction conditions [40,41]. Another study examined the linkage between EO and blood pressure in the general population obtaining several new evidences. Notably: (1) people with the Gly460Trp polymorphism of α-adducin have higher EO plasmatic levels compared to the carriers of the wild-type genotype; (2) the EO plasmatic concentration is directly proportional to urinary potassium excretion; and (3) there is an important interaction between blood pressure, EO levels and urinary sodium excretion [42]. We can finally affirm that EO acts as a positive regulator of blood pressure during chronic low-salt diet, whilst it prevents high salt-induced blood pressure when the salt intake is elevated. There is a correlation between EO and genetics in the homeostatic regulation of blood pressure in response to changes in salt intake (Figure 2). However, this complex relationship requires further investigation in order to be fully clarified.

**Figure 1.** Effect of α-adducin of Na+/K+-ATPase pump. The mutated form of α-adducin reduces endocytosis, leading to an over-expression of the Na+/K+ pump molecules on the basolateral membrane and to an increased sodium reabsorption. In the basal condition, the association between phosphatase A2 (PPA2) and adducin is reduced in tubular cells transfected with mutated adducin. The impairment of this cycle may represent the molecular mechanism underlying the reduced endocytosis observed in the presence of mutated adducin (figure adapted from Bianchi et al., Hypertnsion 2005) [3].

**Figure 2.** Physio-pathological interaction between Endogenous Ouabain (EO) and of α-adducin. This interaction can lead to the development of hypertension and organ maladaptive remodeling and potential target of an anti-ouabain compound (as rostafuroxin) (figure adapted from Ferrandi et al., JBC 2004) [43].
