**4. The Correlation between Endogenous Ouabain and Organ Damage**

## *4.1. Endogenous Ouabain and Cardiovascular Disease*

Studies on rat (normotensive, MNS and MHS rats) and human models have demonstrated that EO contributes not only to the pathogenesis of hypertension, but also to the development of cardiac complications, such as ventricular hypertrophy, heart failure and acute myocardial infarction [12–14]. Approximately half of patients affected by essential hypertension have high circulating levels of EO, and previous studies demonstrated a direct relation between increased EO levels, left ventricular mass indices and stroke volumes, whilst a negative correlation of increased EO levels with heart rates was seen [15–17]. EO might be actually considered as a growth-promoting hormone. Indeed, many authors showed that EO is involved in pro-hypertrophic and pro-fibrotic pathways that lead to cardiac remodeling with a negative impact on both cardiac structures and cardiovascular functions [30,44]. The authors also tried to understand how EO might perform the role of a signal transducer. Studies on cultured vascular cells showed that EO, after the binding to its receptor (Na+/K+-ATPase), could trigger a tyrosine-kinase protein starting an intracellular-signaling cascade that constitutes a stimulus for the epidermal growth factor receptor (EGFR). This pathway finally permits the transcription of gene encoding for pro-fibrotic factors [45], promoting cardiac hypertrophy. Relevant evidences showed that a chronic activation of this complex protein-kinase cascade could finally lead to heart failure [46]. It was hypothesized that the molecular pathway that leads to organ hypertrophy in vivo was similar to the one described in cultured vascular cells [16]. In a study conducted on more than 800 patients undergoing elective cardiac surgery, it was supposed that EO might be also used as a valuable biomarker of heart failure. This study confirmed the already hypothesized negative relation that intercourses between increased EO levels and left ventricular ejection fraction and the positive correlation between EO levels and cardiac end-diastolic diameters. EO were dosed in all of these patients after and before the surgery, and it was seen that higher EO circulating levels both in the pre-operation and the immediate post-operation were associated with worst cardiovascular presentation, higher morbidity and increased risks of perioperative mortality after cardiac surgery [47].

#### *4.2. Endogenous Ouabain and Renal Disease*

The main actor in cardiac glucosides' metabolism is the liver; however, it is now clear that kidneys also have an important role in their clearance. To validate this evidence, experiments conducted on the rat demonstrated that partial nephrectomy was associated with higher EO circulating levels [12]. Similarly, it was seen that the progression of kidney disease, in particular the end-stage renal failure, was associated with an increase of EO levels [19]. High concentrations of EO are comparable to an excessive and not controllable digitalization that might lead to a vasopressor effect and other important cardiac side effects. Indeed, studies on patients with Chronic Kidney Disease (CKD) or undergoing dialysis demonstrated that elevated levels of EO were strongly associated with alterations in ventricular mass and geometry [48,49], independently from blood pressure and other determinants of left ventricular hypertrophy. The comorbidities that characterize patients with severe renal diseases and dialyzed patients, such as hypertension and cardiac hypertrophy, might thus be potentially related to EO. These data stimulated the interest of the impact of EO on the renal function. To understand this aspect, a selective podocyte marker protein called nephrin was used. Studies on rats showed that a prolonged exposure to high levels of EO was associated with a less expression of nephrin in the podocyte, a reduction in creatinine clearance and increased proteinuria. This last finding was also replicated ex vivo with the incubation of low dose of ouabain in podocyte primary cell cultures [50]. EO modulates the Na+/K+-ATPase that is involved in tubular ischemic damage, and it is responsible for the initial nephrinuria and the glomerular damage. The EO's promotion of kidneys' damage shown in rat models suggested a possible role of EO in acute kidney injury (AKI). In a recent observational study, a blood sample for the dosage of EO was taken during the induction of anesthesia in patients destined to elective cardiac surgery. Interestingly, patients with higher preoperative levels of EO

were characterized by worst renal outcomes and higher mortality rates [47,50,51]. Starting from these evidences, it is a fair assumption that EO might be considered to be a valuable biomarker of individual susceptibility to the development of AKI after cardiac surgery [52,53].

Recent studies individuated an additional effect of EO, showing that it might also act as a pro-cystogenic agent in the development of autosomal dominant polycystic kidney disease (ADPKD). It was shown that the Na+/K+-ATPase of ADPKD cells has an increased affinity with EO, and even if the mechanism undergoing this abnormal affinity remains uncertain, it might enhance ADPKD cell's susceptibility to circulating EO. The exposure of primary cultures of cells isolated from renal cysts of ADPKD patients to nanomolar concentrations of EO provokes the proliferation of cyst epithelial cells. In contrast, similar concentrations of EO had a pour influence on the proliferation of normal human kidney cells [52,53].

#### *4.3. Endogenous Ouabain and Brain Disease*

The presence of EO inside the central nervous system (CNS) is well-known since the early 1990s [54,55]. It was demonstrated that it is an integrated component of a hypothalamic renin-angiotensin-aldosterone system (RAAS) and also plays an important role in regulation of systemic Blood Pressure (BP) [37,40,56]. The relation between the central and peripheral RAASes with central and peripheral EO is still not completely understood [55], but it was discovered that EO-induced signaling in neurons had positive and direct consequences on rat brain in terms of brain cells survival [57]. Similarly, in mouse models, the reduction of endogenous steroids seems to have a protective effect on oxidative stress for CNS [58]. Endogenous steroids, in particular EO-like compounds, were also described as potential risk factors involved in the etiology of bipolar disorder [59], mania [60] and depression [61]. Moreover, the use of anti-ouabain antibodies showed a reduction of maniac [60], as well as depressive [62] status. The development of EO associated behavior disorders seems directly associated with a dys-regulation of Na+/K+-ATPase inside CNSes [59,60].
