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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.

Abstract-We have observed recently that experimental renal failure in the rat is accompanied by increases in circulating concentrations of the cardiotonic steroid, marinobufagenin (MBG), and substantial cardiac fibrosis. We performed the following studies to examine whether MBG might directly stimulate cardiac fibroblast collagen production. In vivo studies were performed using the 5/6th nephrectomy model of experimental renal failure (PNx), MBG infusion (MBG), PNx after immunization against MBG, and concomitant PNx and adrenalectomy. Physiological measurements with a Millar catheter and immunohistochemistry were performed. In vitro studies were then pursued with cultured isolated cardiac fibroblasts. We observed that PNx and MBG increased MBG levels, blood pressure, heart size, impaired diastolic function, and caused cardiac fibrosis. PNx after immunization against MBG and concomitant PNx and adrenalectomy had similar blood pressure as PNx but less cardiac hypertrophy, diastolic dysfunction, and cardiac fibrosis. MBG induced increases in procollagen-1 expression by cultured cardiac fibroblasts at 1 nM concentration. These increases in procollagen expression were accompanied by increases in collagen translation and increases in procollagen-1 mRNA without any demonstrable increase in procollagen-1 protein stability. The stimulation of fibroblasts with MBG could be prevented by administration of inhibitors of tyrosine phosphorylation, Src activation, epidermal growth factor receptor transactivation, and N-acetyl cysteine. Based on these findings, we propose that MBG directly induces increases in collagen expression by fibroblasts, and we suggest that this may be important in the cardiac fibrosis seen with experimental renal failure. (Hypertension. 2007;49:215-224.)

Background-Digitalis-like sodium pump ligands (SPLs) effect natriuresis via inhibition of renal tubular Na ϩ ,K ϩ -ATPase but may induce vasoconstriction. The present study investigated the potential roles of 2 putative endogenous SPLs, an ouabain-like compound (OLC) and an ␣ 1 Na ϩ ,K ϩ -ATPase inhibitor, marinobufagenin (MBG), in regulating natriuresis and blood pressure (BP) responses to sustained and acute NaCl loading in Dahl salt-sensitive rats (DS). Methods and Results-During 4 weeks of an 8% NaCl diet, DS exhibited a progressive increase in MBG renal excretion (66Ϯ13 pmol/24 hours at week 4 versus 11Ϯ1 pmol/24 hours at baseline, nϭ48), which paralleled an increase in systolic BP (174Ϯ10 mm Hg at week 4 versus 110Ϯ2 mm Hg at baseline). By contrast, OLC excretion peaked at week 1 and returned to baseline levels. Administration of an anti-MBG, but not anti-ouabain antibody, to DS after 3 weeks of a high NaCl diet lowered BP (139Ϯ7 versus 175Ϯ5 mm Hg, PϽ0.001, nϭ5). Acute NaCl loading (2 hours) of DS (nϭ5) increased MBG and OLC excretion and natriuresis. Pretreatment of acutely NaCl-loaded DS with an anti-MBG antibody (nϭ5) reduced the excretion of sodium and MBG but not that of OLC. An anti-ouabain antibody (nϭ5) reduced sodium excretion and both OLC and MBG. Key Words: hypertension Ⅲ Na ϩ -K ϩ -exchanging ATPase Ⅲ sodium Ⅲ bufadienolides Ⅲ ouabain T he role of endogenous digitalis-like sodium pump ligands (SPLs) in the pathogenesis of hypertension has been disputed for almost 3 decades. 1 Endogenous SPLs are believed to promote natriuresis via renal Na ϩ pump inhibition and may link the Na ϩ retention that occurs during NaCl loading to an increase in arterial pressure via inhibition of the Na ϩ ,K ϩ -ATPase in cardiovascular tissues. [2][3][4] An endogenous ouabain-like compound (OLC) was the first mammalian SPL to be purified. 5 Subsequent studies showed that mammals also produce another class of SPLs, ie, bufadienolides. 6 -8 Bufadienolides, which were discovered in Amphibia, inhibit Na ϩ ,K ϩ -ATPase and cross-react with digitalis antibodies. 9 In Amphibia, skin bufadienolides are responsible for Na ϩ excretion, 9 and bufadienolide levels in toads vary with changes in environmental salinity. 10 Several bufadienolides have been suggested as candidate SPLs in mammals, including marinobufagenin (MBG), 7 which acts in vitro as a vasoconstrictor 11,12 and exhibits greater affinity to rodent ouabain-resistant ␣ 1 isoform of the Na ϩ ,K ϩ -ATPase than to an ␣ 3 isoform. 11,13,14 MBG immunoreactive material purified from human and rat urine was found to be identical to MBG from the toad, Bufo marinus, by mass spectral analysis 15 and by its ability to inhibit rat kidney Na ϩ ,K ϩ -ATPase. 14 Enhanced MBG production occurs in volume-expanded dogs 16 and rats 17 and in pathological states associated with fluid retention, including preeclampsia, 12 hypertension in endstage renal disease, 18 and salt-sensitive hypertension in Dahl rats. 14 In NaCl-induced hypertension in Dahl salt-sensitive rats (DS), which exhibit...

Our observations demonstrate the coexistence of two endogenous cardiotonic steroids in preeclamptic plasma, a more polar OLC and a less polar MBG-like compound. Substantial increases in plasma OLC and MBG immunoreactivity in preeclampsia, along with the vasoconstrictor properties of authentic MBG and Na+,K+ ATPase inhibitory activity of human MBG immunoreactive factor, suggest, that in preeclampsia, plasma concentrations of MBG are enough to substantially inhibit the sodium pump in cardiovascular tissues, and are in accordance with the views attributing endogenous digitalis-like factors a pathogenic role in the preeclamptic hypertension.

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.

The cardiotonic steroid marinobufagenin (MBG) has been implicated in the pathogenesis of experimental uremic cardiomyopathy, which is characterized by progressive cardiac fibrosis. We examined whether the transcription factor Friend leukemia integration-1 (Fli-1) might be involved in this process. Fli-1-knockdown mice demonstrated greater cardiac collagen-1 expression and fibrosis compared with wild-type mice; both developed increased cardiac collagen expression and fibrosis after 5/6 nephrectomy. There was a strong inverse relationship between the expressions of Fli-1 and procollagen in primary culture of rat cardiac and human dermal fibroblasts as well as a cell line derived from renal fibroblasts and MBG-induced decreases in nuclear Fli-1 as well as increases in procollagen-1 expression in these cells. Transfection of a Fli-1 expression vector prevented increased procollagen-1 expression from MBG. MBG exposure induced a rapid translocation of the δ-isoform of protein kinase C (PKCδ) to the nucleus. This translocation was prevented by pharmacological inhibition of phospholipase C, and MBG-induced increases in procollagen-1 expression were prevented with a PKCδ- but not a PKCα-specific inhibitor. Finally, immunoprecipitation studies strongly suggest that MBG induced phosphorylation of Fli-1. We feel these data support a causal relationship with MBG-induced translocation of PKCδ, which results in phosphorylation of as well as decreases in nuclear Fli-1 expression, which, in turn, leads to increases in collagen production. Should these findings be confirmed, we speculate that this pathway may represent a therapeutic target for uremic cardiomyopathy as well as other conditions associated with excessive fibrosis.

Because of the plethora of genetic manipulations available in the mouse, we performed a partial nephrectomy in the mouse and examined whether the phenotypical features of uremic cardiomyopathy described in humans and rats were also present in the murine model. A 5/6 nephrectomy was performed using a combination of electrocautory to decrease renal mass on the left kidney and right surgical nephrectomy. This procedure produced substantial and persistent hypertension as well as increases in circulating concentrations of marinobufagenin. Invasive physiological measurements of cardiac function demonstrated that the 5/6 nephrectomy resulted in impairment of both active and passive left ventricular relaxation at 4 wk whereas tissue Doppler imaging detected changes in diastolic function after 6 wk. Morphologically, hearts demonstrated enlargement and progressive fibrosis, and biochemical measurements demonstrated downregulation of the sarcoplasmic reticulum calcium ATPase as well as increases in collagen-1, fibronectin, and vimentin expression. Our results suggest that partial nephrectomy in the mouse establishes a model of uremic cardiomyopathy which shares phenotypical features with the rat model as well as patients with chronic renal failure.