Plasma Levels Of Marinobufagenin Research Articles

Marinobufagenin, Left Ventricular Hypertrophy and Residual Renal Function in Kidney Transplant Recipients.

Left ventricular hypertrophy (LVH), which is a pervasive complication of end-stage kidney disease (ESKD), persists in some uremic individuals even after kidney transplantation (Ktx), contributing to worsening CV outcomes. Marinobufagenin (MBG), an endogenous steroid cardiotonic hormone endowed with natriuretic and vasoconstrictive properties, is an acknowledged trigger of uremic cardiomyopathy. However, its clinical significance in the setting of Ktx remains undefined. In a cohort of chronic Ktx recipients (n = 40), we assessed circulating MBG together with a thorough clinical and echocardiographic examination. Forty matched haemodialysis (HD) patients and thirty healthy subjects served as controls for MBG measurements. Patients were then prospectively followed up to 12 months and the occurrence of an established cardio-renal endpoint (death, CV events, renal events, graft rejection) was recorded. Median MBG plasma levels were lower in Ktx as compared with HD patients (p = 0.02), but higher as compared with healthy controls (p = 0.0005). Urinary sodium (β = 0.423; p = 0.01) and eGFR (β = -0.324; p = 0.02) were the sole independent predictors of MBG in this cohort, while a strong correlation with left ventricular mass index (LVMi), found in univariate analyses (R = 0.543; p = 0.0007), gained significance only in multivariate models not including eGFR. Logistic regression analyses indicated MBG as a significant predictor of the combined endpoint (OR 2.38 [1.10-5.12] per each 1 nmoL/L increase; p = 0.01), as well as eGFR, LVMi, serum phosphate and proteinuria. Ktx recipients display altered MBG levels which are influenced by sodium balance, renal impairment and the severity of LVH. Thus, MBG might represent an important missing link between reduced graft function and pathological cardiac remodelling and may hold important prognostic value for improving cardio-renal risk assessment.

The importance of antiangiogenic substances endoglin and sFLT-1, as well as endogenous digitalis-like factor marinobufagenin in the pathogenesis of preeclampsia

Objective. Establish the relationship between the action of antiangiogenic factors sFLT-1 and endoglin, as well as marinobufagenin in the formation of symptoms of preeclampsia in the clinic and experiment.
 Materials and methods. In the first experimental phase, preeclampsia-like state was simulated in pregnant rats, the changes in the content of substances reflected in the target tissues were studied, as well as the effect on their concentrations of anti-marinobufagenin antibodies. In the second (clinical) phase, changes in the content of sFlt-1 and endoglin-1 in placental tissues, as well as marinobufagenin in blood plasma and activity of Na+/K+-ATPase of erythrocytes in pregnant women with preeclampsia were studied.
 Results. In rats, an increase in systolic blood pressure, and marinobufagenin plasma levels was observed during the formation of a preeclampsia-like condition. Administration of antibodies to marinobufagenin caused a decrease in blood pressure. Found that during the formation of a preeclampsia-like condition, there is an increase in the content of sFlt-1 in the placenta and thoracic aorta and endoglin in the placenta. In patients with preeclampsia, it was found that the increase in blood pressure occurs against the background of an increase in the content of marinobufagenin in blood plasma, as well as a decrease in the activity of Na+/K+-ATPase of erythrocytes. The formation of preeclampsia is accompanied by a significant increase in the level of antiangiogenic factors endoglin and sFlt-1 in the placenta.
 Summary. In patients with preeclampsia, the development of clinical symptoms is accompanied by an increase in the placental tissues of the content of antiangiogenic factors endoglin and sFlt-1 and the content of marinobufagenin in blood plasma. The obtained data are confirmed by the results of an experiment performed on pregnant rats with modeling of preeclampsia-like condition.

Acute salt loading and cardiotonic steroids in resistant hypertension.

The study addresses the association of marinobufagenin (MBG), a natriuretic and vasoconstrictor steroid, and Na/K-ATPase (NKA) activity with pressor response to salt-loading and arterial stiffness in resistant hypertension (RH). Thirty-four patients (18 males and 16 females; 56±8 years) with RH on a combined (lisnopril/amlodipine/hydrochlorothiazide) therapy and 11 healthy age-matched normotensive subjects (7 males and 4 females; 54±2 years) were enrolled in this study. Salt-loading was performed via intravenous infusion of 1000mL saline (0.9% NaCl) for 1h. Arterial stiffness was measured by Sphygmocor Px device with a calculation of pulse-wave velocity (PWV). Activity of NKA was measured in erythrocytes. We demonstrated that plasma levels of MBG and magnitude of NaCl-induced MBG-dependent NKA inhibition are associated with PWV, and that this association has gender- and age-specific fashion in RH patients.

Low to Normal Plasma Levels of Marinobufagenin 24 Hours or More after an Ischemic Stroke: A Pilot Study.

Numerous studies have demonstrated a strong relationship between circulating levels of marinobufagenin (MBG) and salt-sensitivity. Since salt-sensitive hypertensives have increased plasma levels of MBG and are known to be at a higher risk of having cardiovascular events, stroke and increased mortality, we evaluated the possibility of an association between MBG and ischemic stroke. In this pilot study, we determined plasma MBG levels in patients after surviving an ischemic stroke compared to similar age and gender groups of treated hypertensives and normotensive controls. We measured plasma MBG levels in a total of 40 participants subdivided into three groups: After an ischemic stroke STR (n = 13), participants with a diagnosis of hypertension receiving blood pressure medication HT (n = 14) and normotensive control subjects CTL (n = 13). We used inferential statistics (parametric or non-parametric) and ordered logistic regression models (unadjusted and adjusted) and all statistical analyses were performed using Stata 14. We did not include a subject from the CTL group because of a diagnosis of glucose-6-phosphate dehydrogenase deficiency and an extreme plasma MBG value of 2,246 pmol/L. Participants' mean age was 60.4 ± 11.5 years; 56% were male. There was no significant difference between study groups (p > 0.05) for gender, age, and body mass index. HbA1c levels were significantly higher in the STR as compared to the CTL p < 0.05). In the STR group MBG levels were below the normal range (< 200 pmol/L) in three (23%), eight (61%) were in the normal range (200-400 pmol/L), while two (16%) had increased MBG values (> 400 pmol/L). Also, among the STR, the plasma MBG levels did not differ between those receiving and not receiving thrombolytic therapy (p > 0.05). From the 14 HT participants, six (43%) had MBG plasma levels within the normal range, and eight (57%) had high concentrations (> 400 pmol/L). Four (29%) of the treated hypertensives had extreme MBG levels (> 1,000 pmol/L) and normal values of blood pressure. There was no significant elevation of plasma MBG in survivors 24 h or more after an ischemic stroke. The extreme values of plasma MBG in 29% of the treated hypertensives suggests the presence of salt-sensitivity and a possible side effect of a specific combination of medications. Both of these findings contribute new knowledge to the design of studies to define if there is an MBG molecular mechanism underlying the complex associations among salt-sensitivity, hypertension, and ischemic stroke.

Revealing of endogenous Marinobufagin by an ultra-specific and sensitive UHPLC-MS/MS assay in pregnant women

Marinobufagenin (MBG) is a bufadienolide cardiac inotrope implicated in volume expansion-mediated hypertensive states including essential hypertension and preeclampsia (PE). Endogenous MBG is an inhibitor of the α1-isoform of Na+,K+-ATPase with vasoconstrictive and cardiotonic properties, causing hypertension and natriuresis.Elevated endogenous MBG-like material levels have been described by immunoassays in salt-sensitive pregnant and preeclamptic rats as well as in preeclamptic human patients. The rise of endogenous MBG-like material appears prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential biomarkers for PE.The weak specificity and the high variability of the published immunoassays gives no certification about endogenous MBG existence. This led us to set-up a highly specific and sensitive analytical method to detect MBG in plasma at low levels relying on liquid chromatography combined to mass spectrometry (UHPLC-MS/MS) with recording of 7 highly specific MRM transitions for MBG. Pure MBG standard used in the method development was obtained by purification from the Bufo marinus toad venom. d3–25-hydroxyvitamin D3 was used as internal standard. An increasing organic gradient with mobile phase A and B composed of 97:3 (v/v) H2O: MeOH and 50:45:5 (v/v/v) MeOH:IPA:H2O at pH 4.5 respectively was used on a Pursuit 3 PFP column (100 mm × 3 mm; 3 µm) to allow elution and separation of the plasmatic compounds. Chromatographic analyses of plasma samples were preceded by a precipitation of proteins pretreatment.The developed UHPLC-MS/MS assay has been applied to early-pregnant women plasma samples allowing us to investigate MBG plasma levels. Thanks to the high specificity of the assay we were able to authenticate and certify the presence of endogenous MBG in early-pregnant women plasma with the use of the 7 selected specific mass transitions. These pioneering preliminary results are giving a promising perspective for early preeclampsia risk assessment in pregnant women.

D3. Early prediction of preeclampsia risk assessment: analytical determination for marinobufagenin in pregnant women

Introduction: Marinobufagenin (MBG), an endogenous bufadienolide cardiac inotrope, is responsible for vasoconstriction and natriuresis [1,2]. Growing interest by its implication in volume expansion mediated hypertensive states such as essential hypertension, congestive heart failure, chronic renal failure, primary aldosterism and preeclampsia (PE). Elevated endogenous MBG levels have been described early in preeclamptic patients [2–5].Aims: To develop a specific and sensitive analytical MBG assay with special attention for the limit of quantification (LOQ). An algorithm dealing with the MBG plasma levels might be established in the future, in order to help for prediction of the risk for preeclampsia in pregnant women.Method: A LC-MS/MS based assay designed to determine MBG in human plasma is being optimized. The obtained LOQ fully satisfies the need for quantification of MBG plasma levels in pregnancy (nmol/L range). After a validation process, a primary observational clinical study will allow us to confirm previous results observed in preeclampsia.Results and discussion: We had the opportunity to authenticate the presence of MBG by LC-MS/MS in non-pregnant and in early-pregnant women. These pioneering preliminary results that are giving a promising perspective for early preeclampsia risk assessment in pregnant women will be confirmed with the clinical study.

Cardiotonic steroids: main effects, therapeutic applications

For last few decades 'putative natriuretic hormone' have been considered as very important therapeutic target for developing novel therapies for cardiovascular diseases. The present review discusses the pathophysiological role of endogenous cardiotonic steroids with main focus on marinobufagenin (MBG). Recent studies has established that MBG plays a vital role in regulation of electrolyte homeostasis in humans and rodents. Additionally, it has been reported that elevated MBG plasma levels are associated with number of pathological states such as arterial hypertension, chronic kidney disease, preeclampsia and heart failure. It has been demonstrated that MBG-Na/K-ATPase interaction in kidneys regulates renal sodium excretion inducing natriuresis. Further, it has been reported that MBG-Na/K-ATPase interaction in vascular smooth muscle cells could induce vasoconstriction and cardiovascular fibrosis. Thus these facts have established MBG as a potential therapeutic target. Several therapies such as immunoneutralization of MBG with specific monoclonal antibodies and antagonism with aldosterone antagonists have already been proposed. Further studies providing understanding of pathophysiological implications of MBG and signaling pathways could contribute in establishing new therapies for cardiovascular diseases.

Elevated Plasma Marinobufagenin, An Endogenous Cardiotonic Steroid, Is Associated With Right Ventricular Dysfunction and Nitrative Stress in Heart Failure.

Plasma levels of cardiotonic steroids are elevated in volume-expanded states, such as chronic kidney disease, but the role of these natriuretic hormones in subjects with heart failure (HF) is unclear. We sought to determine the prognostic role of the cardiotonic steroids marinobufagenin (MBG) in HF, particularly in relation to long-term outcomes. We first measured plasma MBG levels and performed comprehensive clinical, laboratory, and echocardiographic assessment in 245 patients with HF. All-cause mortality, cardiac transplantation, and HF hospitalization were tracked for 5 years. In our study cohort, median (interquartile range) MBG was 583 (383-812) pM. Higher MBG was associated with higher myeloperoxidase (r=0.42, P<0.0001), B-type natriuretic peptide (r=0.25, P=0.001), and asymmetrical dimethylarginine (r=0.32, P<0.001). Elevated levels of MBG were associated with measures of worse right ventricular function (RV s', r=-0.39, P<0.0001) and predicted increased risk of adverse clinical outcomes (MBG≥574 pmol/L: hazard ratio 1.58 [1.10-2.31], P=0.014) even after adjustment for age, sex, diabetes mellitus, and ischemic pathogenesis. In mice, a left anterior descending coronary artery ligation model of HF lead to increases in MBG, whereas infusion of MBG into mice for 4 weeks lead to significant increases in myeloperoxidase, asymmetrical dimethylarginine, and cardiac fibrosis. In the setting of HF, elevated plasma levels of MBG are associated with right ventricular dysfunction and predict worse long-term clinical outcomes in multivariable models adjusting for established clinical and biochemical risk factors. Infusion of MBG seems to directly contribute to increased nitrative stress and cardiac fibrosis.

O16. Marinobufagenin as a promising preeclampsia risk assessment marker: purification from toad venom and LC–MS identification in human plasma

Introduction Marinobufagenin (MBG) is an endogenous bufadienolide cardiac inotrope which is demonstrating growing interest in the early diagnosis of volume expansion-mediated hypertensive states such as preeclampsia (PE) and end-stage renal disease hypertension. Mammalian MBG is an inhibitor of the α 1 isoform of Na + , K + -ATPase with vasoconstrictive and cardiotonic properties, resulting in hypertension and natriuresis. Elevated endogenous MBG levels have been described in pregnant mammals and especially in preeclamptic patients [1,2,3]. The rise of endogenous MBG appears prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential target in the biomarker panel for PE. A sensitive and accurate analytical method is needed to assess MBG in as lower level as possible in plasma. Currently, only marinobufagenin-like material has been found in humans using two published quantification methods based each on immunoassays [4,5]. These techniques suffer from a lack of specificity due to cross-reactivity and tend to exhibit high variability at low concentrations [6]. Objective Our aim is to develop a MBG assay using a more specific and easy to access technique, such as LC–MS/MS. An algorithm dealing with the MBG plasma levels might be established by clinicians in the future, in order to predict, in combination with other clinical and biological markers, the risk for preeclampsia in pregnant women. Methods As the major source for MBG is located in the parotid glands of the Bufo Marinus toad, we developed a purification method from toad venom in order to get pure MBG standard. A pre-extraction procedure was elaborated to concentrate and clean the plasma sample prior to its analysis. A LC–MS based assay designed to determine MBG in human plasma is being optimized, giving us the opportunity to investigate MBG in non-pregnant healthy volunteers plasma as well as in early pregnant women plasma. Results and discussion Pure MBG has been successfully extracted from the B. Marinus toad venom and the identity of the compound has been confirmed. An extraction procedure for MBG from plasma has been set up by use of solid phase extraction cartridges. Preliminary results allowed us to authenticate the presence of MBG by LC–MS/MS in non-pregnant women as well as in early pregnancy. Further optimization and validation of the LC–MS assay are needed to quantify MBG plasma levels. However, these pioneering observations, are giving the clinicians a promising perspective for early preeclampsia risk assessment in pregnant women.

СARDIOTONIC STEROIDS NEUTRALIZATION AS A THERAPEUTIC APPROACH IN PREECLAMPSIA

Preeclampsia (PE) is a serious complication of late pregnancy. O bjective . To study and to explore the possibilities of neutralization of cardiotonic steroids, to define the possible mechanisms of recovery activity Na/K-ATPase in pregnant women with PE for the prevention of vasospasm. Design and methods. The study was carried out in two stages. Firstly, two groups of pregnant women were included. Control group consisted of 6 women with normal pregnancy with gestational age 37–40–3/7 weeks. The main group consisted of 7 women with PE with a comparable gestational age (mean systolic blood pressure 157 ±5 mm Hg, diastolic blood pressure 94 ±2 mm Hg, and urinary protein excretion 2,12 ± 0,46 g/day). At the second stage, 12 patients with PE (aged 29 ± 1 year, gestation 37,9 ± 0,6 weeks, blood pressure 159 ± 5 / 99 ±3 mm Hg) formed the main group. The control group included 11 healthy pregnant women comparable by gestational age. Venous blood samples were taken. Marinobufagenin level was determined by fluoroimmunoassay, spectrophotometry was used to assess the activity of erythrocyte Na/K-ATPase in the presence and absence of monoclonal antibody, DigiFab, magnesium sulfate. R esults. The level of marinobufagenin in pregnant women with preeclampsia was almost 2,5-fold higher than in control group (1,056 vs. 0,421 nM). The activity of erythrocyte Na/K-ATPase in pregnant women with preeclampsia was 1,47 ± 0,1716 and 2,65 ± 0,16 mcmol Fn/mL/h (p < 0,01), respectively. When incubated with monoclonal antibodies erythrocyte Na/K–ATPase activity was 2,41 mcmol Fn/mL/h. When incubated with DigiFab there was a 3,5-fold increase in PE marinobufagenin plasma levels (1,38 ± 0,40 vs. 0,38 ± 0,10 nmol/L, p < 0,01) and a reduction of the erythrocyte Na/K–ATPase activity compared to control group (1,16 ± 0,11 vs. 2,80 ± 0,2 mkmol Fn/ml/h, p < 0,01). Ex vivo, 1 ug/ml DigiFab restores the activity of Na/K–ATPase (1,72 ± 0,13 mkmol Fn/ml/h, p < 0,01) and3 mM magnesium sulfate amplifies DigiFab effect (2,3 ± 0,2 mkmol Fn/mL/h, p < 0,01).

PP089. Analytical aspects of marinobufagenin and its applications in the diagnosis of preeclampsia.

Marinobufagenin (MBG), a bufadienolide cardiac inotrope, enjoys a growing interest in the early diagnosis of volume expansion-mediated hypertensive states such as preeclampsia (PE). This endogenous mammalian vasoconstrictive compound, is a selective inhibitor of the α1 subunit of Na+,K+-ATPase, leading to hypertension and natriuresis. Enhanced production of MBG has been described in preeclamptic patients prior the development of hypertension and proteinuria, leading to consider MBG as a biomarker for PE [1-3]. However, the role of MBG as a biomarker remains to be fully understood as well as his biosynthetic pathway. The need has arisen for an accuracy and sensitive analytical method of MBG plasma levels in order to further investigate the implications of MBG in PE, and to help to establish a diagnosis for this syndrome. Our aim is to develop a sensitive and robust analytical MBG dosage method allowing quantifications as low as possible. A critical threshold value may be established in order to discriminate normal pregnant from preeclamptic women. Nowadays, the MBG standard compound is not commercially available. It forced us to develop an extraction method of MBG from the crystallized toad Bufo Marinus venom. A pre-extraction step on rat and human plasma is performed in order to clean and to concentrate samples. Several liquid chromatography (LC) strategies coupled with different detection methods are considered and performed. Pure MBG has been successfully extracted from the crystallized toad venom. The identity of the compound has been confirmed by different spectral techniques. The pre-extraction step from plasma samples spiked with MBG has been carried out through a solid phase extraction HLB cartridge with an extraction yield of 88%. Preliminary reversed-phase LC method allows quantifications of MBG between 6μg/mL and 11μg/mL.

Interaction of Digibind with endogenous cardiotonic steroids from preeclamptic placentae

Preeclampsia is a major cause of maternal and fetal mortality, and its pathogenesis is not fully understood. Endogenous digitalis-like cardiotonic steroids (CTS) have been implicated in the pathophysiology of preeclampsia; this is illustrated by clinical observations that Digibind, a therapeutic digoxin antibody fragment which binds CTS, lowers blood pressure and reverses Na/K-ATPase inhibition in patients with preeclampsia. Recently we reported that plasma levels of marinobufagenin (MBG), a bufadienolide vasoconstrictor CTS, are increased four-fold in patients with severe preeclampsia. In the present study, we compared levels of MBG in normal and preeclamptic placentae, as well as the interactions of Digibind and antibodies against MBG and ouabain with material purified from preeclamptic placentae using high-performance liquid chromatography (HPLC). Levels of endogenous MBG, but not that of endogenous ouabain, exhibited a four-fold elevation in preeclamptic placentae vs. normal placentae (13.6 +/- 2.5 and 48.6 +/- 7.0 nmoles/g tissue; P < 0.01). The elution time of endogenous placental MBG-like immunoreactive material from reverse-phase HPLC column was identical to that of authentic MBG. A competitive immunoassay based on Digibind exhibited reactivity to HPLC fractions having retention times similar to that seen with MBG and other bufadienolides, but not to ouabain-like immunoreactive material. Our results suggest that elevated levels of endogenous bufadienolide CTS represent a potential target for immunoneutralization in patients with preeclampsia.

Marinobufagenin causes endothelial cell monolayer hyperpermeability by altering apoptotic signaling

Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid that is involved in the inhibition of the sodium pump Na(+)/K(+)-ATPase. Increased plasma levels of MBG have been reported in patients with preeclampsia. MBG increases microvascular barrier permeability in an animal model of preeclampsia. However, the mechanism by which MBG impairs endothelial permeability is unknown. We utilized rat lung microvascular endothelial cells (RLMEC) to examine alterations in MBG-induced monolayer permeability and the effect of MBG on the phosphorylation status of ERK1/2, Jnk, and p38. Apoptosis was evaluated by examining alterations in caspases 3/7, 8, and 9 and annexin-V staining. We also examined the effect of MBG on the endothelial adherens junctions of the RLMEC monolayer. MBG inhibited the proliferation, and increased the monolayer permeability, of RLMEC. These actions of MBG were attenuated by ERK, p38, and pan caspase inhibition. MBG significantly decreased the phosphorylation of ERK1/2 and activated the phosphorylation of Jnk and p38. MBG also significantly increased the expression of caspases 3/7, 8, and 9, indicating the activation of apoptosis. MBG-induced apoptosis signaling was not observed in cells pretreated with a p38 inhibitor. MBG treatment induced the disruption of endothelial cell junctions. This effect was prevented by a pan caspase inhibitor. In conclusion, 1) MBG induced an impairment of RLMEC proliferation; 2) the bufadienolide also caused endothelial hyperpermeability; and 3) these effects of MBG were mediated by the downregulation of ERK1/2, the upregulation of Jnk and p38, by the activation of apoptosis, and by the disruption of endothelial cell junctions.

Immunoneutralization of endogenousNa/K-ATPase inhibitors in preeclampsia

Although preeclampsia (PE) is a major cause of maternal and fetal mortality, its pathogenesis is not fully understood. Endogenous digitalis-like cardiotonic steroids (CTS) are implicated in the pathophysiology of PE, as illustrated by clinical observations that Digibind, a digoxin antibody which binds CTS, lowers blood pressure in patients with PE. Recently we reported that plasma levels of marinobufagenin (MBG), a bufadienolide vasoconstrictor CTS, are increased four-fold in patients with severe PE. In the present study, we compared levels of MBG in normal and preeclamptic placentae, and tested whether antibodies against MBG, and against ouabain for their interaction with the material purified from preeclamptic placentae via high-performance liquid chromatography (HPLC). Levels of MBG, but not that of endogenous ouabain, exhibited a four-fold elevation in preeclamptic placentae. The elution time of endogenous placental MBG-like immunoreactive material from reverse-phase HPLC column was identical to that of authentic MBG. Immunoassay based on Digibind did not detect cross-reactivity with HPLC containing ouabain-like immunoreactive material, but cross-reacted with HPLC fractions having retention time similar to that of MBG and other bufadienolides. Our results demonstrate that levels of MBG are significantly elevated in preeclamptic placentae, and suggest that MBG is a potential target for immunoneutralization in patients with preeclampsia.

Marinobufagenin Inhibits Proliferation and Migration of Cytotrophoblast and CHO Cells

Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid that is involved in the inhibition of the sodium pump Na +/K +-ATPase. Increased plasma levels of MBG have been reported in patients with volume expansion-mediated hypertension and preeclampsia. We have recently demonstrated that MBG impairs both the proliferation and growth factor-induced migration of human first trimester cytotrophoblast (CTB) cells, crucial for proper placental development. However, the intracellular signaling mechanisms regulating the MBG-induced impairment of CTB differentiation, migration and invasion are unknown. The human extravillous CTB cell line SGHPL-4 was utilized for this study. The phosphorylation of MAP kinase protein ERK1/2 was evaluated by Cellular Activation of Signaling ELISA (CASE) in control CTB cells and those treated with MBG. MBG at concentrations of 10 and 100 nM inhibited CTB cell proliferation, migration and invasion (60%, 50% and 50%, respectively). MBG also caused a significant decrease in the phosphorylation of ERK1/2. In addition, MBG decreased proliferation, migration, and ERK1/2 activity in another motile cell line, CHO cells. Another sodium pump inhibitor, ouabain, similarly decreased proliferation and ERK1/2 activity in CTB and CHO cells. These data suggest that the changes observed in cell function may be mediated by inhibition of Na +/K +-ATPase. We demonstrate that the MBG-induced impairment of CTB cell proliferation, migration and invasion is associated with decreased ERK1/2 activity which may be mediated by inhibition of Na +/K +-ATPase.

Endogenous Na/K-ATPase inhibitors in patients with preeclampsia.

Although preeclampsia (PE) is a major cause of maternal and fetal mortality, its pathogenesis is not fully understood. Digitalis-like cardiotonic steroids (CTS) are believed to be involved in the pathophysiology of PE, as illustrated by clinical observations that DIGIBIND, a digoxin antibody which binds CTS, lowers blood pressure in PE. Recently we reported that plasma levels of marinobufagenin (MBG), a vasoconstrictor CTS, are increased fourfold in patients with severe PE. In the present study, we tested whether anti-MBG, or anti-ouabain antibodies, or DIGIBIND can reverse inhibition of erythrocyte Na/K-ATPase (NKA) from patients with mild PE (blood pressure, 149 +/- 3/93 +/- 3 mm Hg; age, 28 +/- 2 years; gestational age, 37 +/- 1 weeks). Development of PE was associated with twofold rise in plasma MBG levels (1.58 +/- 0.15 vs. 0.80 +/- 0.11 nmol/L; P<0.01). The activity of erythrocyte NKA in 12 patients with PE was lower than in 6 normotensive gestational age-matched subjects (1.56 +/- 0.18 vs. 3.11 +/- 0.16 micromol Pi/ml/hr; P<0.001). In vitro treatment of erythrocytes from PE patients with anti-MBG antibody fully restored the NKA activity (3.26 +/- 0.41 micromol Pi/ml/hr; P<0.01). The effects of DIGIBIND was marginally significant (2.53 +/- 0.32 micromol Pi/ml/hr), while the anti-ouabain antibody was not effective (2.25 +/- 0.25 micromol Pi/ml/hr, P>0.5). The present observations provide evidence for a role for MBG in the pathogenesis of PE, and suggest that antibodies against MBG may be useful in the treatment of this syndrome.

Marinobufagenin, an endogenous Na/K-ATPase inhibitor, in preeclampsia

Although preeclampsia (PE) is a major cause of maternal and fetal mortality, its pathogenesis is not fully understood. Endogenous digitalis-like sodium pump ligands (SPLs) are believed to be involved in pathophysiology of PE, as illustrated by clinical observations that DIGIBIND, a digoxin antibody which binds SPLs. Lowers blood pressure (BP) in PE. Recently we reported that plasma levels of marinobufagenin (MBG), a vasoconstrictor SPL, are increased four-fold in patients with severe PE. In the present study, we tested whether polyclonal antibodies to MBG and ouabain, and DIGIBIND can reverse inhibition of erythrocyte Na/K-ATPase (NKN) from patients with mild PE. Development of PE was associated with two-told rise in plasma MBG levels (1.58 ± 0.15 vs. 0.80 ± 0.11 nmol/E: P 0.5) were less effective. Our present observations provide evidence for a role of MBG in pathogenesis of PE, and suggest that antibodies against MBG may be used to treat the syndrome.

Antibody to marinobufagenin lowers blood pressure in pregnant rats on a high NaCl intake

The pathogenesis of pre-eclampsia (PE), a major cause of maternal and fetal mortality, is not fully understood. Digitalis-like sodium pump ligands (SPLs) are believed to be implicated in PE, as illustrated by clinical observations that DIGIBIND, a digoxin antibody that binds SPLs, lowers blood pressure (BP) in PE. We recently reported that plasma levels of marinobufagenin (MBG), a vasoconstrictor SPL, are increased four-fold in patients with PE. In the present study, we tested whether a polyclonal antibody to MBG can lower BP in rats with pregnancy-associated hypertension. Systolic BP (SBP), 24-h renal excretion of MBG and endogenous ouabain (EO), and sodium pump activity in the thoracic aortae were measured in virgin and pregnant Sprague-Dawley rats without and with NaCl supplementation (drinking 1.8% NaCl solution). NaCl supplementation of virgin rats stimulated renal excretion of MBG by 60%, but not that of EO, and did not change the BP. Compared with virgin rats, the last week of pregnancy in non-NaCl-loaded rats was associated with a decrease in SBP (106 +/- 2 versus 117 +/- 2 mmHg); a moderate increase in renal excretion of MBG (97.6 +/- 4.9 versus 57.4 +/- 7.0 pmoles/24 h) and EO (36.2 +/- 4.3 versus 24.1 +/- 3.2 pmoles/24 h). NaCl-loaded pregnant rats exhibited elevation in SBP (139 +/- 3 mmHg; P < 0.01 versus non-NaCl-loaded pregnant rats), in renal excretion of MBG (160.0 +/- 17.5 pmoles/24 h; P < 0.01 versus non-NaCl-loaded pregnant rats), but not in EO, and showed fetal growth retardation. Administration of the anti-MBG antibody to NaCl-loaded pregnant rats lowered SBP (111 +/- 2 mmHg; P < 0.01) and increased aortic sodium pump activity (144 +/- 3 versus 113 +/- 5 nmol Rb/g per min; P < 0.01 versus non-NaCl-loaded pregnant rats). These observations provide evidence that MBG contributes to BP elevation in pregnant rats rendered hypertensive by NaCl supplementation.

Endogenous digitalis-like ligands and Na/K-ATPase inhibition in experimental diabetes mellitus

Dysregulation of the Na/K-ATPase (NKA) in the kidney, cardiovascular system, and peripheral nervous system is believed to contribute to pathogenesis of diabetes mellitus (DM) and its complications. Recently we demonstrated that, in addition to endogenous ouabain (EO), mammalian tissues contain another NKA inhibitor, a bufadienolide marinobufagenin (MBG). In vitro MBG, a natriuretic and a vasoconstrictor, acts as a selective inhibitor of alpha-1 NKA, the main isoform of the sodium pump in renal tubules and vascular smooth muscle. To determine whether digitalis-like NKA inhibitors are linked to NKA dysregulation in DM, we studied changes in renal excretion and plasma levels of MBG and EO and the activity of erythrocyte NKA in male Wistar rats with type 1 DM and type 2 DM. Rats with type 1 DM were studied four weeks following a single intraperitoneal injection of 65 mg/kg streptozotocin (STZ)(n = 12), and rats with type 2 DM were studied 10 weeks after intraperitoneal injection of STZ during the neonatal period (n = 12). Renal excretion and plasma levels of EO did not change in rats with both types of DM as compared to that in the control groups. Renal excretion (57.5 +/- 9.4 pmol/kg/ 3 hours vs. 12.6 +/- 2.1 pmol/kg/ 3 hours; P less than 0.01) and plasma levels (2.23 +/- 0.82 nmol/L vs. 0.29 +/- 0.07 nmol/L; P less than 0.01) of MBG increased, and NKA activity in erythrocytes was inhibited by 50% in rats with type 1 DM as compared to controls. In rats with type 2 DM, plasma levels (1.48 +/- 0.09 nmol/L vs. 0.46 +/- 0.02 nmol/L; P less than 0.01) and renal excretion (21.3 +/- 3.2 pmol/kg/ 3 hours vs. 13.1 +/- 2.1 pmol/kg/ 3 hours) of MBG also became elevated, but less than in the animals with type 1 DM. Accordingly, activity of NKA in erythrocytes from rats with type 2 DM was inhibited by 35%. In vitro treatment of erythrocytes from rats with type 1 and type 2 DM with anti-MBG antibody reversed the DM induced inhibition of the NKA. These results suggest that digitalis-like factors are involved in the pathogenesis of DM and that MBG, rather than EO, is responsible for DM-induced NKA inhibition.

Marinobufagenin, an endogenous ligand of alpha-1 sodium pump, is a marker of congestive heart failure severity.

A reduced cardiac output in chronic heart failure (CHF) evokes renal NaCl and water retention, and, therefore, activates mechanisms promoting natriuresis. Atrial natriuretic peptide (ANP) is one such factor. We hypothesized that another NaCl sensitive endogenous natriuretic factor, i.e., marinobufagenin (MBG), a specific ligand of the alpha-1 subunit of Na/K ATPase (the main kidney isoform) and also a vasoconstrictor and cardiotonic substance, would be elevated in CHF patients in a graded manner with the severity of CHF. We measured the plasma levels of MBG, alpha-hANP, ouabain-like compound (OLC) and left ventricular (LV) volumes and ejection fraction in 23 consecutive hypertensive male patients with CHF. Plasma MBG levels exhibited progressive increases (0.59 +/- 0.15, 1.08 +/- 0.20, 1.35 +/- 0.17 and 1.88 +/- 0.05 nmol/l NYHA 1-4, respectively) and paralleled the changes of alpha-hANP. Conversely, plasma OLC did not exhibit such increases. Plasma MBG correlated with alpha-hANP (r = 0.82; P < 0.0001). Both MBG and alpha-hANP correlated with LV systolic (r = 0.55 and r = 0.47; P < 0.01) diameter and inversely with ejection fraction (r = -0.73 and r = -0.60; P < 0.01). OLC did not exhibit correlations with alpha-hANP or LV volumes, but positively correlated with systolic brachial blood pressure and with pulse pressure. In CHF, MBG exhibits progressive increases similar to ANP, varies with CHF severity and correlates with LV systolic function. We hypothesize, that, in CHF, the concurrent production of these two natriuretic hormones, a vasorelaxant, ANP, and a vasoconstrictor, MBG, potentiate each other's natriuretic effects, but may offset their vasoactive actions.