**5. Conclusions**

Endogenous circulating CTS such as ouabain and MBG are known to be upregulated in the body's stress response towards volume expansion. Binding to its receptor—the Na/K-ATPase —leads to increased sodium excretion in the proximal tubules of the nephron. Whether this is

accomplished through the classic ionic pathway, the alternate signaling pathway, or both is still debated; however, its effect on re-establishing volume homeostasis is undeniable. Like many other physiological processes, the fine-tuning of one pathway may result in unintended consequences elsewhere in the body. In the case of CTS-induced natriuresis through Na/K-ATPase signaling, the tradeoff is apparent in the development of cardiac and renal fibrosis as demonstrated both in vivo and in vitro (Figure 2). Because the fibrosis appears to be dependent on Na/K-ATPase signaling, the generation of ROS, and subsequent oxidative stress to cardiac and renal tissues, this creates the potential for both new and old drugs to target and block the signaling cascade. ROS scavengers, Src inhibitors, spironolactone, and canrenone have already demonstrated exciting possibilities in our experiments. Further research in developing more specific CTS antagonists as well as whether this concept can be extrapolated to humans needs to be explored. Interestingly, in the late 1990's and early 2000's, Pitt and colleagues conducted the Randomized Aldactone Evaluation Study (RALES), followed by the Eplerenone Post– Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS) and determined that spironolactone and eplerenone, respectively, were cardioprotective in patients with advanced stages of congestive heart failure [76, 77]. More recently, the Eplere‐ none in Mild Patients Hospitalization and Survival Study in Heart Failure (EMPHASIS-HF) and the Anti-Remodeling Effect of Canrenone in Patients with Mild Chronic Heart Failure (AREA IN-CHF) trials found further therapeutic benefit in patients with mild (NYHA Class II) CHF [78, 79]. These clinical studies proposed that the anti-aldosterone effects were primarily responsible for the reduction in morbidity and mortality; however, it is certainly plausible to speculate whether these drugs as Na/K-ATPase signaling inhibitors may have also played a role. Nonetheless, CTS-induced signaling through the Na/K-ATPase is a significant and novel link in balancing the hemodynamics of salt handling and the development of fibrosis. Signaling Pathway: Relationship Between Fibrosis and Natriuresis

was significantly reduced in both partial nephrectomy and MBG-infusion experimental

In a separate set of experiments, Wansapura subjected genetically altered ouabain-sensitive mice (originally developed by Lingrel) to aortic banding in order to simulate a pressure overload model. After four weeks, the ouabain-sensitive group was noted to have developed substantially greater cardiac hypertrophy and fibrosis compared to ouabain-resistant (wildtype) mice. Furthermore, the administration of Digibind to the ouabain-sensitive mice

Additional in-vivo data support the potential for a more significant reversal of cardiotonic steroid associated hypertension, cardiac hypertrophy and oxidative stress through utilization of a monoclonal antibody (mAb). Haller subjected partially nephrectomized rats demonstrat‐ ing elevated levels of MBG to mAb with a high affinity for MBG and Digibind. Both treatments resulted in significant improvement in cardiac hypertrophy, hypertension and measures of oxidative stress. While both therapies demonstrated similar responses there were significantly

Furthermore, in cultures of rat cardiac and renal fibroblasts as well as human dermal fibro‐ blasts, we found that both ouabain and MBG were able to directly increase collagen production and proline incorporation [67, 71, 72]. By inducing a translocation of PKC to the nucleus, MBG appears to cause the subsequent phosphorylation and degradation of Friend leukemia integration-1 (Fli-1), which Watson and colleagues have demonstrated is a negative regulator of collagen synthesis in dermal fibroblasts [71, 73]. In fact, we found MBG reduced Fli-1 expression and increased procollagen-1 in all three of our fibroblast cell lines (cardiac, renal, dermal) [71]. Furthermore, MBG infusion stimulates the expression and nuclear translocation of Snail, a transcription factor involved in epithelial-mesenchymal transition, which is

Similar to the animal studies, these findings in cell culture have corresponded with increased Na/K-ATPase signaling activity and the generation of ROS, which notably was successfully blocked by both ROS scavengers and Src inhibitors [67]. In addition, we examined the effects of spironolactone—known to be a competitive antagonist of CTS binding to the Na/K-ATPase —as well as its major metabolite, canrenone. Corroborating with our hypothesis, we found that spironolactone significantly attenuated cardiac fibrosis in the renal failure models, and both spironolactone and canrenone reduced collagen production in cardiac fibroblasts. It was further demonstrated that MBG-induced Na/K-ATPase signaling was blocked in these

Endogenous circulating CTS such as ouabain and MBG are known to be upregulated in the body's stress response towards volume expansion. Binding to its receptor—the Na/K-ATPase —leads to increased sodium excretion in the proximal tubules of the nephron. Whether this is

groups [2, 66-68].

86 Cardiomyopathies

diminished these cardiac changes [69].

implicated in renal fibrosis [74].

experiments [75].

**5. Conclusions**

better results with the MGG directed monoclonal antibody [70].

**Figure 2.** Signaling Pathway: Relationship Between Fibrosis and Natriuresis
