Abstract-Patients with chronic renal failure develop a "uremic" cardiomyopathy characterized by diastolic dysfunction, cardiac hypertrophy, and systemic oxidant stress. Patients with chronic renal failure are also known to have increases in the circulating concentrations of the cardiotonic steroid marinobufagenin (MBG). On this background, we hypothesized that elevations in circulating MBG may be involved in the cardiomyopathy. First, we observed that administration of MBG (10 g/kg per day) for 4 weeks caused comparable increases in plasma MBG as partial nephrectomy at 4 weeks. MBG infusion caused increases in conscious blood pressure, cardiac weight, and the time constant for left ventricular relaxation similar to partial nephrectomy. Decreases in the expression of the cardiac sarcoplasmic reticulum ATPase, cardiac fibrosis, and systemic oxidant stress were observed with both MBG infusion and partial nephrectomy. Next, rats were actively immunized against a MBG-BSA conjugate or BSA control, and partial nephrectomy was subsequently performed. Immunization against MBG attenuated the cardiac hypertrophy, impairment of diastolic function, cardiac fibrosis, and systemic oxidant stress seen with partial nephrectomy without a significant effect on conscious blood pressure. These data suggest that the increased concentrations of MBG are important in the cardiac disease and oxidant stress state seen with renal failure.

Background Levels of marinobufagenin (MBG), an endogenous bufadienolide Na/K-ATPase (NKA) inhibitor, increase in preeclampsia and in NaCl-sensitive hypertension. Methods We tested a 3E9 monoclonal anti-MBG antibody (mAb) for the ability to lower blood pressure (BP) in NaCl-sensitive hypertension and to reverse the preeclampsia-induced inhibition of erythrocyte NKA. Measurements of MBG were performed via immunoassay based on 4G4 anti-MBG mAb. Results In hypertensive Dahl-S rats, an intraperitoneal administration of 50 μg/kg 3E9 mAb lowered BP by 40 mmHg and activated Na/K-pump in thoracic aorta by 51%. NaCl supplementation of pregnant rats (n = 16) produced a 37 mmHg increase in BP, a 3.5-fold rise in MBG excretion, and a 25% inhibition of the Na/K-pump in the thoracic aorta, compared with pregnant rats on a normal NaCl intake. In eight pregnant hypertensive rats, 3E9 mAb reduced the BP (25 mmHg) and restored the vascular Na/K-pump. In 14 patients with preeclampsia (mean BP, 126 ± 3 mmHg; 26.9 ± 1.4 years; gestational age, 37 ± 0.8 weeks), plasma MBG was increased three-fold and erythrocyte NKA was inhibited compared with that of 12 normotensive pregnant women (mean BP, 71 W 3 mmHg)(1.5 ± 0.1 vs. 3.1 ± 0.2 μmol Pi/ml/h, respectively; P < .01). Ex-vivo 3E9 mAb restored NKA activity in erythrocytes from patients with preeclampsia. As compared with 3E9 mAb, Digibind, an affinity-purified antidigoxin antibody, was less active with respect to lowering BP in both hypertensive models and to restoration of NKA from erythrocytes from patients with preeclampsia. Conclusion Anti-MBG mAbs may be a useful tool in the studies of MBG in vitro and in vivo and may offer treatment of preeclampsia.

These observations provide evidence that MBG contributes to BP elevation in pregnant rats rendered hypertensive by NaCl supplementation.

Abstract-Dahl salt-sensitive rats (DS), which have a mutation in the ␣-1 subunit of Na ϩ /K ϩ -ATPase, exhibit impaired pressure natriuresis and on a high-salt diet, retain Na ϩ and exhibit increased blood pressure. Recently, we have shown that mammalian tissues contain a bufadienolide Na ϩ /K ϩ -ATPase inhibitory factor, marinobufagenin (MBG), that exhibits greater affinity for the ␣-1 than ␣-3 sodium pump isoform. The present study investigated the possible role of MBG in hypertension in DS on a high NaCl intake. Eight DS and 8 Dahl salt-resistant rats (DR) were placed on an 8% NaCl diet. Within 2 weeks, systolic blood pressure increased in DS (162Ϯ9 mm Hg at week 2 versus 110Ϯ2 mm Hg in baseline, PϽ0.01), and increased less in DR (124Ϯ3 mm Hg at week 2 versus 112Ϯ2 mm Hg in baseline). Renal excretion of MBG increased 4-fold (38.9Ϯ7.6 pmol versus 9.1Ϯ1.3 pmol in baseline, PϽ0.01) in DS, but by only 25% in DR (13.2Ϯ0.9 pmol versus 10.3Ϯ0.7 pmol in baseline). Excretion of endogenous ouabain did not change in either strain. MBG-immunoreactive material was purified from the urine of hypertensive DS by means of 2 steps of reverse-phase high performance liquid chromatography (HPLC) and compared with plant ouabain and amphibian MBG for its ability to inhibit the Na ϩ /K ϩ -ATPase from rat kidney (which expresses only ␣-1 Na ϩ /K ϩ -ATPase isoform). Unlike ouabain (IC 50 ϭ248 mol/L), serially diluted, HPLC-purified MBG immunoreactivity from DS and authentic MBG potently inhibited rat kidney Na ϩ /K ϩ -ATPase (IC 50 ϭ70 and 78 nmol/L, respectively). Our results suggest that an ␣-1 Na ϩ /K ϩ -ATPase ligand, MBG, is elaborated to promote natriuresis in hypertensive DS. MBG acts as a selective inhibitor of the ouabain-resistant ␣-1 Na ϩ /K ϩ -ATPase subunit, ie, the major sodium pump isoform of the kidneys, as would be expected of a putative natriuretic hormone. Key Words: NaCl Ⅲ Dahl rats Ⅲ Na ϩ /K ϩ -ATPase Ⅲ kidney Ⅲ bufadienolides Ⅲ marinobufagenin T he Dahl-deWardener-Blaustein concept of natriuretic hormone postulates that in volume-expanded forms of hypertension, an enhanced production of endogenous digitalis-like sodium pump ligand (SPL) occurs with a primarily adaptive aim, to decrease the volume of circulating fluid by means of inhibition of the Na ϩ /K ϩ -ATPase in renal tubules. The excessive SPL production also contributes to hypertension by means of inhibition of the Na ϩ /K ϩ -ATPase in cardiovascular tissues. 1-3 Several SPL have been described in mammalian tissues, including a cardenolide, ouabain-like compound (OLC), 4,5 and bufadienolides, 6 -8 including marinobufagenin (MBG) -immunoreactive factor. 9 MBG (3␤,5␤-dihydroxy-14,15-epoxybufadienolide) was originally discovered in amphibia. 10 Subsequently, MBG immunoreactive material purified from human urine exhibited mass-spectral properties identical to those of amphibian MBG. 11 In contrast to ouabain, MBG exhibits a greater affinity for the ouabainresistant ␣-1 subunit of Na ϩ /K ϩ -ATPase 12,13 , the main sodium pump isoform in renal tubules. In vitro...