**4. Marinobufagenin and Chronic Kidney Disease**

CKD is one of the most frequent medical conditions worldwide and, in the general population, the CKD prevalence of all five KDIGO stages is 13.4% [84]. Low estimated glomerular filtration rate (eGFR) is a strong, independent predictor of all-cause mortality and CV diseases [85], which are the first cause of morbidity and mortality in nephropathic patients. Their increased CV risk is related to both traditional (diabetes mellitus, hypertension, etc.) and non-traditional uremia-specific risk factors [86–88]. Given the high social, economic and health impact of CKD worldwide, new possible underlying pathogenetic mechanisms and potential markers that may allow the early identification of the development of this complex and multifaceted disease are always being investigated.

It has been shown that, in both animal models (rats and dogs) [5,89] and in humans [74] with volume-expanding conditions, there are high plasma concentration levels of MBG. Indeed, it has been widely demonstrated that MBG production increases in all conditions of sodium and fluid retention, such as essential hypertension, heart failure, pre-eclampsia (PE), salt-sensitive hypertension in Dahl salt-sensitive (DS) rats on a high NaCl intake [46,90] and CKD.

The influence of MBG in CKD and its complications were initially evaluated in animal models. Originally, the involvement of MBG in cardiac hypertrophy was investigated in a remnant model of CKD [6,7]. In a series of investigations of these remnant kidney models, it has been shown that the development of renal dysfunction is associated with an increase in circulating concentrations of MBG [91]. On the other hand, other studies have shown an increased collagen production through fibroblasts and subsequent fibrosis in experimental uremic cardiomyopathy (UC) [9]. UC is characterized by an association with left ventricular hypertrophy (LVH) and myocardial fibrosis [92]. The etiopathogenesis of UC is extremely complex and involves several factors, such as hemodynamic overload, hypertension, anemia, mineral and bone disorders, endothelial dysfunction, insulin resistance and cardiotonic steroids, as well as several circulating uremic toxins [92,93]. It was assumed that the increase in MBG concentration was secondary to renal failure-dependent volume expansion [58]. Although extracellular volume expansion is thought to be crucial for the development of UC, there is still much debate about the exact pathogenesis [94–96]. Immunization against MBG in partial nephrectomy animals was associated with a substantial attenuation of cardiac hypertrophy, cardiac fibrosis and the oxidant stress state [97].

The fibrotic action of MBG on the kidney was also evaluated through the infusion of MBG in rats, which led to a peritubular and periglomerular accumulation of type I collagen at the renal cortical level [61,98]. This could be triggered by the activation of Transforming Growth Factor Beta type 1 (TGF-β1) via the renin–angiotensin–aldosterone system. In MBG-treated kidneys, the profibrotic transcription factor snail (critical regulator of the epithelial–mesenchymal transition) was expressed in both medullary and cortical tubular epithelial cells. This evidence led to a new hypothesis: MBG can have a key causative role in the epithelial–mesenchymal transition [61]. The administration of MRA occupying endogenous CTS-binding sites prevents pro-fibrotic MBG effects [64]. Since CKD is a complex set of different medical conditions, Na+/K+-ATPase alterations may not always be involved in the etiopathogenesis of different causes of CKD and MRA therapy may not be effective in all nephropathic patients. Therefore, increased MBG plasma levels could be useful in identifying patients at risk of developing renal fibrosis (and beyond) and CKD progression that could benefit from MRA therapy [19].

Recently, in a single-center study of adults routinely referred for the screening of endocrine hypertension, it was shown that plasma MBG concentrations were significantly associated with albuminuria (a marker of kidney damage) and decline in renal function regardless of pre-existing CKD. These results might indicate that in this cohort of hypertensive patients, MBG could play a role as a potential marker of renal failure at follow-up and that elevated plasma levels of MBG may already precede renal failure rather than being a simple consequence of it [19].

In patients undergoing chronic hemodialysis, the altered MBG plasma values could be due to a compensatory response to the treatment itself [20] and could predict a worsening survival outcome [21]. In this patient population, left ventricular hypertrophy is extremely prevalent and, as mentioned above, contrasts the uremic cardiomyopathy. In experimental models, MBG induces marked hypertrophic changes of adult cardiac myocytes in vitro [10,99] and promotes vascular fibrosis and cardiac hypertrophy in experimental models of salt-sensitive hypertension [91]. For these reasons, we conducted a pilot observational study to investigate a possible relationship between MBG plasma levels, left ventricular (LV) geometry and cardiac dysfunction in end-stage renal disease (ESRD) patients on dialysis [22]. In this cohort of patients with ESRD, we observed that high levels of MBG reflect the structural and functional alterations of the LV. Indeed, MBG plasma levels were higher in the presence of diastolic dysfunction and this molecule demonstrated a strong diagnostic ability to discern patients with normal LV geometry, LV hypertrophy and, above all, eccentric LVH. Circulating MBG plasma levels were significantly higher in ESRD patients than those in healthy controls and were more increased in patients on peritoneal dialysis compared with those undergoing extracorporeal dialysis treatment [22]. This suggests that, in the future, MBG could play the role of biomarker for cardiac evaluation in high-risk populations. Our findings further strengthen the hypothesis that endogenous cardiotonic steroids could substantially contribute to the onset and progression of uremic cardiomyopathy. Of note, there was no correlation between MBG plasma levels and parameters related to volume status in our study [22]. In a recent study, kidney transplant recipients displayed altered MBG levels, which were influenced by sodium balance, renal impairment and the severity of LVH. Thus, MBG might also represent an important missing link between reduced graft function and pathological cardiac remodeling and may hold important prognostic value for improving cardio-renal risk assessment [100].

Moreover, we have demonstrated that higher MBG plasma levels are associated with a lower risk of intradialytic hypotensive events in patients undergoing hemodialysis [20,101,102], who are particularly considered at risk of hypotension. Indeed, about 30% of hemodialysis sessions are characterized by severe symptomatic intra-dialysis hypotension, influencing the morbidity and mortality of patients in chronic treatment. These hypotensive episodes are often due either to an altered ability to mobilize fluids from the interstitial space to the intravascular space during the hemodialysis session or to a removal of a large fluid volume in a short time. It has been noted that, in the cohort of patients on dialysis treatment, patients with lower baseline MBG values showed an approximately five-fold higher risk of severe symptomatic, intradialytic hypotension. There was an initial increase in circulating MBG levels followed by a progressive decrease until the end of treatment. This shows that patients with lower MBG plasma values reflect a lower vascular and hemodynamic tolerance, with a higher number of episodes of severe hypotension, while no significant correlation was found between MBG plasma levels and body weight reduction during dialysis treatment. This could lead to a consideration of MBG as a mediator of a compensatory mechanism, which results in an altered hemodynamic response to plasma volume reduction. Today, it seems that MBG may have an important role in identifying patients at high risk of severe intradialytic hypotensive episodes; this predictive ability has also been found in survival analyses, demonstrating that patients with lower plasma levels of MBG had a higher risk (from four to six times) of severe intra-dialysis episodes during follow-up. MBG plasma levels were the strongest time-dependent predictors of severe intradialytic hypotensive episodes between different variables [20]. The high mobilization

of MBG could initially represent a protective response against the hemodynamic changes induced by the extracorporeal treatment but, in the long term, could, in any case, determine the known deleterious effects in the myocardium [20] (Figure 1). However, larger studies with more targeted-oriented surveys are necessary to confirm these data.

**Figure 1.** Main known pathways in the interactions between MBG and different organs and systems.
