Abstract 18178: Cardiotonic Steroid Lactone Ring Hydrolysis by Paraoxonases Attenuates Na/K ATPase Mediated Signaling

Abstract

Introduction: Cardiotonic steroids (CTS) such as marinobufagenin and telocinobufagin (TCB) are significantly elevated in chronic kidney disease (CKD) and heart failure and can directly lead to inflammation and cardiac fibrosis upon binding and signaling through the Na+/K+-ATPase. The 17β-lactone ring of CTS is critical for Na+/K+-ATPase binding. Paraoxonases are a family of hydrolytic enzymes which exhibit lactonase activity, although their physiologic substrate(s) are unclear. Decreased serum PON activity and increased levels of CTS are associated with increased oxidant stress and adverse clinical outcomes in the setting of HF and CKD, yet whether decreased PON activity is mechanistically linked to increased CTS levels is unclear. Hypothesis: We tested the hypothesis that PONs are capable of CTS hydrolysis and that hydrolysis of CTS to their open-ring form renders them inactive. Methods/Results: First we measured the ability of recombinant PONs to hydrolyze the lactone ring of active TCB to form open-ring TCB (ORT). Recombinant PON-1 (58.0±2.0 U/mg), PON-2 (0.49±0.01 U/mg) and PON-3 (10.8±0.6 U/mg) had specific lactonase activity for TCB as measured with a continuous pH sensitive colormetric assay. Further, plasma from PON-1 KO mice had a markedly diminished ability to open the 17β-lactone ring of TCB to form resulting ORT aldehyde as measured by a Purpald reagent assay, whereas plasma from PON1-overexpressing mice had increased production of ORT compared to wild type controls. Data from 86-Rubidium uptake experiments in LLC-PK1 cells demonstrated ORT had significantly reduced inhibition of Na+/K+-ATPase compared to TCB (0.25nM of TCB vs. 5000 nM of ORT, p<0.01). In both HK2 and human cardiac fibroblast cells, TCB but not ORT increased collagen-1 mRNA production in a dose dependent manner (1 to 100 nM, p<0.01). Finally, in plasma from CKD patients (n=22) both marinobufagenin (r2=0.58; p<0.01) and TCB (r2=0.31; p<0.01) levels were inversely proportional to lactonase activity, suggesting higher lactonase activity is associated with lower levels of CTS. Conclusion: These studies suggest CTS may be a novel physiologic substrate for PON and represent a previously unknown counter-regulatory mechanism of CTS in the setting of CKD and heart failure.