Adducin- and Ouabain-Related Gene Variants Predict the Antihypertensive Activity of Rostafuroxin, Part 1: Experimental Studies

Abstract

Essential hypertension is a complex, multifactorial disease associated with a high cardiovascular risk and whose genetic-molecular basis is heterogeneous and largely unknown. Although multiple antihypertensive therapies are available, the large individual variability in drug response results in only a modest reduction of the cardiovascular risk and unsatisfactory control of blood pressure in the hypertensive population as a whole. Two mechanisms, among others, are associated with essential hypertension and related organ damage: mutant α-adducin variants and high concentrations of endogenous ouabain. An antihypertensive agent, rostafuroxin, selectively inhibits these mechanisms in rodents. We investigated the molecular and functional effects of mutant α-adducin, ouabain, and rostafuroxin in hypertensive rats, human cells, and cell-free systems and demonstrated that both mutant α-adducin variants and the ouabain-Na,K-ATPase (Na(+)- and K(+)-dependent adenosine triphosphatase) complex can interact with the Src-SH2 (Src homology 2) domain, increasing Src activity and the Src-dependent Na,K-ATPase phosphorylation and activity. Wild-type α-adducin or Na,K-ATPase in the absence of ouabain showed no interaction with the Src-SH2 domain. Rostafuroxin disrupted the interactions between the Src-SH2 domain and mutant α-adducin or the ouabain-Na,K-ATPase complex and blunted Src activation and Na,K-ATPase phosphorylation, resulting in blood pressure normalization in the hypertensive rats. We have also shown the translatability of these data to humans in a pharmacogenomic clinical trial, as described in the companion paper.

Rostafuroxin Protects from Podocyte Injury and Proteinuria Induced by Adducin Genetic Variants and Ouabain

“…Conversely, the NB congenic substrain showed SBP levels similar to those of MNS rats (Table 1) (Tripodi et al, 2004;Ferrandi et al, 2010b). The prolonged oral treatment with rostafuroxin (100 mg/kg per day for 8 weeks, starting from weaning) reduced SBP in MHS and NA rats, but was inactive in MNS rats (Table 1), confirming previous data (Ferrandi et al, 2010a). HR was significantly lower in MHS as compared with MNS rats, and rostafuroxin treatment did not affect HR in any rat strain (Table 1).…”

“…It relies on its ability to inhibit the interaction of mutant a-adducin-or ouabain-activated Na,K-ATPase at the Src-SH2 domain, preventing Src activation and the Srcdependent Na,K-ATPase phosphorylation and activation at the renal tubular level (Ferrandi et al, 2010a). The result is the disruption of the complex signaling cascade downstream to Src-Na,K-ATPase induced by rostafuroxin, which is associated with the normalization of BP in hypertensive rats and patients (Ferrandi et al, 2010a;Lanzani et al, 2010).…”

“…We based our hypothesis on previous findings indicating that rostafuroxin antagonizes mutant a-adducin-and ouabain-activated Na,KATPase interaction at the Src-SH2 domain, resulting in BP normalization through the modulation of renal tubular Na,KATPase activity and its related signaling (Ferrandi et al, , 2010a.…”

“…In this context, we have contributed to unraveling the role of a polymorphism of the cytoskeletal protein adducin and high levels of endogenous ouabain (EO) in hypertension and glomerular podocyte damage, both in animal models and in patients (Bianchi et al, 1994;Bianchi, 2005;Manunta et al, 2009;Ferrandi et al, 2010a,b). These studies have shown that mutant a-adducin and ouabain activate Src tyrosine kinase and the Src-dependent Na,K-ATPase phosphorylation and its signaling network, associated with an overall increase of renal tubular sodium reabsorption and blood pressure (BP) (Ferrandi et al, 2010a). Rostafuroxin, a novel antihypertensive compound (Quadri et al, 1997), disrupts mutant a-adducin-and ouabainmediated interactions, favoring the normalization of renal Src-Na, K-ATPase signaling and BP in hypertensive rats (Ferrari et al, 1998(Ferrari et al, , 1999aFerrandi et al, 2010a) and in patients carrying a specific genetic profile that includes genes encoding for adducin variants and enzymes controlling ouabain synthesis and transport (Lanzani et al, 2010).…”

Do Src Kinase and Caveolin Interact Directly with Na,K-ATPase?

“…Furthermore, an antihypertensive drug, rostafuroxin, exerts its antihypertensive and also antihypertrophic effects by antagonizing the Src-dependent signaling triggered by ouabain (46). In vivo, rostafuroxin acts as an antagonist of endogenous ouabain and has been shown to disrupt an interaction between Src kinase and adducin (47,48). Thus, ouabain may affect the adducin and Src interaction.…”