*Mechanism of Action*

The most studied of the bufadienolides is MBG, an endogenous mammalian natriuretic and cardiotonic compound with vasoconstrictive effects [1,46,47], which has a great affinity for the α1 isoform of Na+/K+-ATPase [48], the main form of the enzyme present in renal tubules [1]. In contrast, cardenolides act primarily on the α2 and α3 isoforms [48]. The Na+/K+-ATPase consists of an alpha subunit with catalytic action together with binding sites for ATP, cardiotonic steroids (CTS) and other ligands, as well as a beta subunit. Four α isoforms and three β isoforms are known for this enzyme. The α1β1 complex is largely present in various tissues, and the α2 isoform is mainly present in cardiac, smooth muscle and cerebral tissues. One of the peculiarities of the bufadienolides is represented by the fact that they exert a different action according to the receptor on which they act [49]. Currently, the Na+/K+-ATPase is recognized as having three major functions: as a pump, as an enzyme and as a receptor to cardiotonic steroids [50]. A "signalling" function has also been recognized, whereby the plasmalemmal Na+/K+-ATPases reside in the caveolae of cells with other key signaling proteins [51,52]. Indeed, two distinct pathways of MBG action have been described, by which MBG acts on the Na+/K+-ATPase [50]. According to the first (defined as the ionic pathway), MBG causes an altered transmembrane ion transport by inhibiting the Na+/K+ ATPase, and this, in the kidney, results in natriuresis as a physiological response to sodium load [4,46,53]. The inhibition of Na+/K+-ATPase in the vascular smooth muscle cells induces vasoconstriction [54,55] through an increase in intracellular sodium concentration and the concomitant reversal of the function of the vascular Na+/Ca++-exchanger. This results in an increased calcium influx within smooth muscle cells, consequently causing the further release of calcium from the sarcoplasmic reticulum. The result is vasoconstriction secondary to the actin–myosin interaction [50,56]. The second mode of action (the signaling function) can cause the activation of several intracellular signals, such as mitogen-activated protein kinases (MAPK) and reactive oxygen species (ROS) inducing fibrosis [50,57]. MBG has been suggested to cause cardiac [57,58]

and vascular [57,59] fibrosis, simply through the activation of the above intracellular signaling cascades.

The inhibition of Na+/K+ ATPase, caused by MBG, has different effects, depending on the tissue in which it occurs. For example, in renal tubules, it stimulates natriuresis and, at the level of the proximal tubule, it promotes the internalization of the sodium pump with a reduction in the expression of the transport protein Na+/H+ (NHE3) in the apical membrane of the renal proximal tubule [60]. MBG, after binding to the enzyme Na+/K+-ATPase, slowly dissociates to induce the endocytosis of this enzyme. This reduces sodium absorption and increases sodium excretion in the proximal renal tubule. By decreasing the amount of Na+/K+-ATPase available, it also decreases the ability to respond to changes in Na<sup>+</sup> and water, leading to the promotion of water retention and volume expansion [61]. In mammals, sodium stimulates the synthesis and secretion of MBG via the angiotensin/sympathetic pathway [34]. Indeed, increased sodium intake promotes angiotensin II, aldosterone and sympathetic nervous system synthesis, resulting in the stimulation of adrenal MBG synthesis and secretion [62].

In healthy young adults, 24 h urinary MBG values were strongly linked with habitual salt intake. This is confirmed by data derived from studies in rats, whose stimulation of MBG through the intake of Na<sup>+</sup> or the infusion of MBG leads to cardiac hypertrophy and vascular fibrosis [24]. In one study, a four-week administration of MBG in rats caused a significant increase in plasma aldosterone and increased systolic blood pressure values. In another study on rats, MBG infusion caused renal fibrosis, subsequently attenuated by passive immunization and improving renal function [59,63]. The use of mineralocorticoid receptor antagonists (MRA) has also been shown to have a preventive effect on MBGinduced fibrosis by occupying the binding sites of the endogenous cardiotonic steroids [64].

With regard to profibrotic pathways, Drummond et al. [65] reported possible opposite relationships between MBG and the antifibrotic microRNA miR-29b-3p, which emerged from the regulation of cardiac fibrosis in a CKD murine model, because of the Na+/K+- ATPase signaling involvement in miR-29b-3p regulation. Similar results were observed in another study conducted in cardiac fibroblasts [66,67], in which cardiac tissue was obtained from rats treated with MBG or partial nephrectomy surgery, showing opposite trends between MBG and miR-29b-3p expression in relation to collagen expression and thereby the extent of fibrosis. All together, these data indicated that CTS mediate the Na+/K+-ATPase signaling-induced regulation of miR-29b-3p expression. The mechanistic phenomena underlying this crosstalk is still yet to be totally defined and are probably due to the convergent action on molecules and kinases common to several pathways.
