Preeclampsia: Cardiotonic Steroids, Fibrosis, Fli1 and Hint to Carcinogenesis.

Despite prophylaxis and attempts to select a therapy, the frequency of preeclampsia does not decrease and it still takes the leading position in the structure of maternal mortality and morbidity worldwide. In this review, we present a new theory of the etiology and pathogenesis of preeclampsia that is based on the interaction of Na/K-ATPase and its endogenous ligands including marinobufagenin. The signaling pathway of marinobufagenin involves an inhibition of transcriptional factor Fli1, a negative regulator of collagen synthesis, followed by the deposition of collagen in the vascular tissues and altered vascular functions. Moreover, in vitro and in vivo neutralization of marinobufagenin is associated with the restoration of Fli1. The inverse relationship between marinobufagenin and Fli1 opens new possibilities in the treatment of cancer; as Fli1 is a proto-oncogene, a hypothesis on the suppression of Fli1 by cardiotonic steroids as a potential anti-tumor therapeutic strategy is discussed as well. We propose a novel therapy of preeclampsia that is based on immunoneutralization of the marinobufagenin by monoclonal antibodies, which is capable of impairing marinobufagenin-Na/K-ATPase interactions.

Evaluation of in vitro neutralization of epoxyscillirosidine by antibodies raised in sheep.

Intoxication by Moraea pallida Bak. (yellow tulp) in livestock is of great importance in South Africa, ranking top among all plant-induced cardiac glycoside toxicosis. The toxic principle, a bufadienolide, is 1α, 2α-epoxyscillirosidine. Treatment of poisoning is challenging and affected livestock often succumbs due to the stress of handling. Manipulating animals to resist poisoning is a potential management strategy. The goal of this study was to explore the potential to develop a vaccine against epoxyscillirosidine by raising antibodies against epoxyscillirosidine in sheep and to assess the neutralization ability of the antibodies in vitro. Epoxyscillirosidine was successfully conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA) rendering them immunogenic. The sheep, vaccinated with epoxyscillirosidine-KLH conjugate (n = 4) and KLH (n = 2) with Montanide, developed antibodies as determined with an indirect enzyme linked immunosorbent assay (ELISA). Total immunoglobulins from sera of vaccinated and control sheep that were purified and concentrated using ammonium sulphate precipitation were 11,940 and 7850 µg, respectively. The in vitro neutralization assay using the methyl blue tetrazolium bromide (MTT) cell viability assay indicated no significant difference (p > 0.05) between anti-epoxyscillirosidine-KLH and KLH antibodies. Rather, the antibodies seemed to enhance the cytotoxicity of epoxyscillirosidine in H9c2 cells. Thus, it is necessary to develop improved vaccination methods to generate antibodies capable of neutralizing the functional group responsible for epoxyscillirosidine toxicity.

Antibody to Marinobufagenin Reverses Placenta-Induced Fibrosis of Umbilical Arteries in Preeclampsia.

BACKGROUND: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Immunoneutralization of heightened MBG by Digibind, a digoxin antibody, reduces blood pressure (BP) in patients with PE, and anti-MBG monoclonal antibody lessens BP in a rat model of PE. Recently, we demonstrated that MBG induces fibrosis in cardiovascular tissues via a mechanism involving inhibition of Fli-1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis. OBJECTIVES AND METHODS: We hypothesized that in PE, elevated placental MBG levels are associated with development of fibrosis in umbilical arteries. Eleven patients with PE (mean BP 124 ± 4 mmHg; age 29 ± 2 years; 39 weeks gest. age) and 10 gestational age-matched normal pregnant subjects (mean BP 92 ± 2 mmHg; controls) were enrolled in the clinical study. RESULTS: PE was associated with a higher placental (0.04 ± 0.01 vs. 0.49 ± 0.11 pmol/g; p < 0.01) and plasma MBG (0.5 ± 0.1 vs. 1.6 ± 0.5 nmol/L; p < 0.01), lower Na/K-ATPase activity in erythrocytes (2.7 ± 0.2 vs. 1.5 ± 0.2 µmol Pi/mL/hr; p < 0.01), 9-fold decrease of Fli-1 level and 2.5-fold increase of collagen-1 in placentae (p < 0.01) vs. control. Incubation of umbilical arteries from control patients with 1 nmol/L MBG was associated with four-fold decrease in Fli-1 level and two-fold increase in collagen-1 level vs. those incubated with placebo (p < 0.01), i.e., physiological concentration of MBG mimicked effect of PE in vitro. Collagen-1 abundance in umbilical arteries from PE patients was 4-fold higher than in control arteries, and this PE-associated fibrosis was reversed by monoclonal anti-MBG antibody ex vivo. CONCLUSION: These results demonstrate that elevated placental MBG level is implicated in the development of fibrosis of the placenta and umbilical arteries in PE.

Aortic Fibrosis, Induced by High Salt Intake in the Absence of Hypertensive Response, is Reduced by a Monoclonal Antibody to Marinobufagenin.

BACKGROUND: Marinobufagenin (MBG) is an endogenous Na/K-ATPase inhibitor, a natriuretic and a vasoconstrictor. MBG is implicated in salt-sensitive hypertension, cardiac hypertrophy, and initiate the pro-fibrotic signaling. Previously it was demonstrated that immunoneutralization of an endogenous MBG by 3E9 anti-MBG-antibody (mAb) in vivo lowered blood pressure (BP) and reversed cardiac fibrosis in salt-sensitive, and in partially nephrectomized rats. In the present study, we investigated whether mAb alleviates vascular remodeling induced in normotensive rats on high salt intake. METHODS: Wistar rats (5 months old) received normal (CTRL; n = 8) or high salt intake (2% NaCl in drinking water) for 4 weeks ( n = 16). Rats from the group on a high salt intake were administered vehicle (SALT; n = 8) or mAb (50 µg/kg) (SALT-AB; n = 8) during the last week of high salt diet. BP, erythrocyte Na/K-ATPase activity, levels of MBG in plasma and 24-hour urine, and sensitivity of aortic explants to the vasorelaxant effect of sodium nitroprusside (SNP) were measured. Aortic collagen abundance was determined immunohistochemically. RESULTS: In SALT vs. CTRL, heightened levels of MBG were associated with inhibition of erythrocyte Na/K-ATPase in the absence of BP changes. High salt intake was accompanied by a 2.5-fold increase in aortic collagen abundance and by a reduction of sensitivity of aortic explants to the vasorelaxant effect of SNP following endothelin-1-induced constriction. In the SALT-AB group, all NaCl-mediated effects were reversed by immunoneutralization of MBG. CONCLUSIONS: High salt intake in young normotensive rats can induce vascular fibrosis via pressure-independent/MBG-dependent mechanisms, and this remodeling is reduced by immunoneutralization of MBG.

Passive immunization against marinobufagenin attenuates renal fibrosis and improves renal function in experimental renal disease.

BACKGROUND: We have shown that the cardiotonic steroid marinobufagenin (MBG) is elevated in clinical and experimental renal disease, and significantly contributes to the development of experimental uremic cardiomyopathy induced by removal of five-sixths of the kidney (5/6 nephrectomy; PNx) in the rat. We have demonstrated that both active and passive immunization against MBG with an anti-MBG monoclonal antibody (mAb 3E9) significantly attenuated cardiac fibrosis following PNx. In the present study we sought to determine whether the use of mAb 3E9 could improve renal function following PNx. METHODS: Sprague-Dawley rats were treated with either mAb 3E9 or with DigiFab (an affinity-purified anti-digoxin antibody formerly named Digibind) during the fourth week after PNx. Sham-operated animals and PNx animals treated with an IgG antibody served as controls. Plasma, urine, and renal tissue were collected at the completion of the study to determine the effects of antibody treatment on renal function. RESULTS: In PNx rats, treatments with mAb 3E9 and DigiFab, respectively, significantly reduced plasma creatinine, improved creatinine clearance, and reduced proteinuria below the values of these three measures in IgG-treated PNx controls. Additionally, treatment with mAb 3E9 and DigiFab significantly reduced renal fibrosis as measured with Western blotting and Sirius red/Fast green staining. CONCLUSIONS: Passive immunization against MBG significantly improved renal function and markedly reduced renal fibrosis following the experimental induction of renal disease. The work in the study reported here adds to a growing body of knowledge implicating MBG in the development of chronic renal disease. Passive immunization against cardiotonic steroids may serve as a promising treatment for chronic renal disease.

DigiFab interacts with endogenous cardiotonic steroids and reverses preeclampsia-induced Na/K-ATPase inhibition.

Elevated levels of endogenous Na/K-ATPase (NKA) inhibitors, cardiotonic steroids (CTSs) including marinobufagenin (MBG), contribute to pathogenesis of preeclampsia (PE) and represent a target for immunoneutralization by Digibind (Ovine Digoxin Immune Antibody, Glaxo-Smith Kline). Because Digibind is no longer commercially available, we studied whether DigiFab (BTG International Ltd, UK) can substitute Digibind for immunoneutralization of CTS in patients with PE. We compared DigiFab, Digibind, and anti-MBG monoclonal antibody (mAb) with respect to their ability to interact with CTS in PE plasma and to restore NKA activity in erythrocytes from patients with PE. Using immunoassays based on DigiFab, Digibind, and anti-MBG mAb, we studied the elution profile of CTS following high-performance liquid chromatography (HPLC) fractionation of PE plasma. Totally, 7 patients with mild PE (28 ± 2 years; gestational age, 39 ± 0.5 weeks; blood pressure 156 ± 5/94 ± 2 mm Hg) and 6 normotensive pregnant participants (28 ± 1 years; gestational age, 39 ± 0.4 weeks; blood pressure 111 ± 2/73 ± 2 mm Hg) were enrolled. Preeclampsia was associated with a substantial inhibition of erythrocyte NKA (1.47 ± 0.17 vs 2.65 ± 0.16 µmol Pi/mL per h in control group, P < .001). Ex vivo, at 10 µg/mL concentration, which is consistent with the clinical dosing of Digibind administered previously in PE, DigiFab and Digibind as well as anti-MBG mAb (0.5 µg/mL) restored erythrocyte NKA activity. Following HPLC fractionation of pooled PE and control plasma, PE-associated increase in CTS material was detected by Digibind (176 vs 75 pmoles), DigiFab (221 vs 70 pmoles), and anti-MBG mAb (1056 vs 421 pmoles). Therefore, because DigiFab interacts with CTS from PE plasma and reverses PE-induced NKA inhibition, it can substitute Digibind for immunoneutralization of CTS in patients with PE.

Monoclonal antibody against marinobufagenin reverses cardiac fibrosis in rats with chronic renal failure.

BACKGROUND: Cardiotonic steroids (CTS) are implicated in pathophysiology of uremic cardiomyopathy. In the present study, we tested whether a monoclonal antibody (mAb) against the bufadienolide CTS, marinobufagenin (MBG), alleviates cardiac hypertrophy and fibrosis in partially nephrectomized (PNx) rats. METHODS: In PNx rats, we compared the effects of 3E9 anti-MBG mAb and of Digibind, an affinity-purified digoxin antibody, on blood pressure and cardiac hypertrophy and fibrosis following 4 weeks after the surgery. RESULTS: In PNx rats, a fourfold elevation in plasma MBG levels was associated with hypertension, increased cardiac levels of carbonylated protein, cardiac hypertrophy, a reduction in cardiac expression of a nuclear transcription factor which is a negative regulator of collagen synthesis, Friend leukemia integration-1 (Fli-1), and an increase in the levels of collagen-1. A single intraperitoneal administration of 3E9 mAb to PNx rats reduced blood pressure by 59 mm Hg for 7 days and produced a significant reduction in cardiac weight and cardiac levels of oxidative stress, an increase in the expression of Fli-1, and a reduction in cardiac fibrosis. The effects of Digibind were similar to those of 3E9 mAb, but were less pronounced. CONCLUSIONS: In experimental chronic renal failure, elevated levels of MBG contribute to hypertension and induce cardiac fibrosis via suppression of Fli-1, representing a potential target for therapy.

[Experimental study of suspicious sensitization component screening on Huachansu injection].

OBJECTIVE: To explore a new experimental method for screening of allergens in post-market traditional Chinese medicine injections by confirming allergens in Huachansu injection. METHOD: First of all, the serum of patients allergic to Huachansu injection were collected, at the same time, the dubious allergen was conjugated to bovine serum albumin (BSA) by EDC coupling procedure to form complete antigen (BNP-BSA), which makes it possible to reproduce the allergic reaction of Huachansu injection in vitro. The histamine liberation ratio, the level of TNF-alpha and Histamine released from RBL-2H3 mast cell were detected; the above data were compared with those obtained in vivo. RESULT: The difference of the histamine liberation ratio, the levels of TNF-alpha and histamine of the resibufogenin-BSA group, group of patients allergic to Huachansu injection were not significant compared with those of normal control group. However, there were significant difference in those data among the cinobufagin-BSA group, the blank control and normal control group (P<0.05). CONCLUSION: The allergen in the serum collected from patients allergic to Huachansu injection is resibufogenin.

Immunomodulatory effects of cinobufagin isolated from Chan Su on activation and cytokines secretion of immunocyte in vitro.

The objective of this study was to evaluate the immunomodulatory effects of cinobufagin (CBG) isolated from Chan Su (Venenum Bufonis) in vitro. In this paper, our results show that CBG significantly stimulated cell proliferation of splenocytes and peritoneal macrophages (PMΦ) and markedly enhanced the phagocytic activation of PMΦ. CBG also significantly increased CD4(+)CD8(+) double-positive T-cell populations and the percentage of S-phase cells of splenic lymphocytes. The levels of several Th1 cytokines, including interferon-γ and tumor necrosis factor-α, are significantly increased after CBG treatment, whereas the levels of the Th2 cytokine interleukin-4 and interleukin-10 are significantly decreased. As a result, the ratio of Th1/Th2 also increased. Taken together, these results indicated that CBG had potential immune system regulatory effects and suggested that this compound could be developed as a novel immunotherapeutic agent to treat immune-mediated diseases such as cancer.

Endogenous cardiotonic steroids in chronic renal failure.

BACKGROUND: Previous reports demonstrated that digitalis-like cardiotonic steroids (CTS) contribute to the pathogenesis of end-stage renal disease. The goal of the present study was to define the nature of CTS in patients with chronic kidney disease (CKD) and in partially nephrectomized (PNx) rats. METHODS: In patients with CKD and in healthy controls, we determined plasma levels of marinobufagenin (MBG) and endogenous ouabain (EO) and erythrocyte Na/K-ATPase activity in the absence and in the presence of 3E9 anti-MBG monoclonal antibody (mAb) and Digibind. Levels of MBG and EO were also determined in sham-operated Sprague-Dawley rats and in rats following 4 weeks of PNx. RESULTS: In 25 patients with CKD plasma, MBG but not EO was increased (0.86 ± 0.07 versus 0.28 ± 0.02 nmol/L, P < 0.01) and erythrocyte Na/K-ATPase was inhibited (1.24 ± 0.10 versus 2.80 ± 0.09 µmol Pi/mL/h, P < 0.01) as compared to that in 19 healthy subjects. Ex vivo, 3E9 mAb restored Na/K-ATPase in erythrocytes from patients with CKD but did not affect Na/K-ATPase from control subjects. Following chromatographic fractionation of uremic versus normal plasma, a competitive immunoassay based on anti-MBG mAb detected a 3-fold increase in the level of endogenous material having retention time similar to that seen with MBG. A similar pattern of CTS changes was observed in uremic rats. As compared to sham-operated animals, PNx rats exhibited 3-fold elevated levels of MBG but not that of EO. CONCLUSIONS: In chronic renal failure, elevated levels of a bufadienolide CTS, MBG, contribute to Na/K-ATPase inhibition and may represent a potential target for therapy.

A chemifluorescent immunoassay for the determination of marinobufagenin in body fluids.

We describe here the development of a chemifluorescent competitive enzyme-linked immunosorbent assay (ELISA) that quantifies marinobufagenin (MBG) levels in biological fluids. Based on a polyclonal antibody raised against a novel MBG-bovine serum albumin conjugate, this assay achieved an MBG detection limit of less than 9 pg/mL. MBG levels in various rat urine and serum samples were effectively determined using this methodology. Interassay variability averaged 9.8%, while intra-assay variability averaged 1.9 and 2.5% in representative serum and urine samples, respectively. Recovery of exogenously added MBG averaged 106%, and parallelism data further established the accuracy of the assay. Employment of this assay to detect MBG abnormalities represents a powerful tool for the possible diagnosis, prevention and management of human hypertensive states, particularly preeclampsia.

Interaction of Digibind with endogenous cardiotonic steroids from preeclamptic placentae.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: Our results suggest that elevated levels of endogenous bufadienolide CTS represent a potential target for immunoneutralization in patients with preeclampsia.

Monoclonal antibody to an endogenous bufadienolide, marinobufagenin, reverses preeclampsia-induced Na/K-ATPase inhibition and lowers blood pressure in NaCl-sensitive 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, intraperitoneal administration of 50 microg/kg 3E9 mAb lowered BP by 32 mmHg and activated the Na/K-pump in the 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 (21 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 +/- 3 mmHg) (1.5 +/- 0.1 vs. 3.1 +/- 0.2 micromol Pi/ml/h, respectively; P < 0.01). 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 studies of MBG in vitro and in vivo and may offer treatment of preeclampsia.

Comparison of non-digitalis binding properties of digoxin-specific Fabs using direct binding methods.

Digibind and DigiFab are commercial formulations of polyclonal, ovine, digoxin-specific Fabs in clinical use for treatment of digoxin intoxication. Of interest for extending its use to other clinical indications, Digibind has also been reported to neutralize the effect of endogenous digoxin-like molecules, including ouabain, that are linked to clinical disorders ranging from preeclampsia to congestive heart failure. Although Digibind and DigiFab are equivalent in their digoxin-binding activity, the antigens used to produce these Fabs are different. We therefore explored, using native (3)H-digoxin and (3)H-ouabain in four different types of solution-phase binding methods, whether they might exhibit different profiles with respect to ouabain and other digoxin-like factors. Consistent with previous results, both Fab preparations bound digoxin with the same affinities and capacities. However, (3)H-ouabain was found to bind with high affinity only to Fab sub-populations present in both products. Interestingly, this sub-population was twice as large for Digibind compared to DigiFab. Competition experiments also showed differences in specificity within Fab sub-populations. Therefore, the equivalence in digoxin-binding activity of the two Fab preparations does not extend to ouabain-binding capacity and Fab specificity, with implications for clinical differentiation between the preparations in treatment of disorders related to control of non-digoxin cardenolides. The existence of a small but perhaps clinically relevant sub-population of antibodies was detected using specific radioligands. This sub-population could not have been detected nor quantified using standard cross-reactivity in an ELISA assay.

Novel digitalis-like factor, marinobufotoxin, isolated from cultured Y-1 cells, and its hypertensive effect in rats.

Marinobufagenin and telecinobufagin have been identified as digitalis-like factors in mammals. In toads, marinobufagenin-related compounds, such as marinobufotoxin (MBT), have been isolated in some tissues but not in mammals, and its biological action has not been elucidated. Herein, we aimed to explore the possible production and/or secretion of MBT and the biological action in rats. First, the MBT in culture supernatant of the adrenocortical-originated cell line Y-1 was analyzed by high-performance liquid chromatography and sensitive ELISA for marinobufagenin-like immunoreactivity. Moreover, the structural information was obtained by mass spectrometry. To determine the biological action, MBT (9.6 and 0.96 microg/kg per day) was intraperitoneally infused via an osmotic minipump for 1 week. Blood pressure and renal excretion of marinobufagenin-like immunoreactivity were measured. Marinobufagenin-like immunoreactivity was found in Y-1 cell culture media, and the concentration increased until 24 hours. The structural analysis suggested that marinobufagenin-like immunoreactivities were marinobufagenin and MBT, and tandem mass spectrum analysis revealed them with the specific daughter ions. The highest sensitive ELISA-positive peak of marinobufagenin-like immunoreactivity in the media was MBT. Continuous administration of MBT in rats for 1 week significantly increased systolic blood pressure and renal excretion of marinobufagenin-like immunoreactivity compared with control rats (135+/-3.0 versus 126+/-2.0 mm Hg and 1.41+/-0.286 versus 0.34+/-0.064 ng/day, respectively). These data suggest that MBT, arginine-suberoyl ester of marinobufagenin, can be a novel digitalis-like factor with hypertensive action and is secreted from the adrenocortical cells.

A potential krimpsiekte vaccine.

Krimpsiekte, a chronic form of cardiac glycoside poisoning, is an important plant-induced intoxication of small stock in South Africa. It is caused by cumulative, neurotoxic bufadienolides, such as cotyledoside. A cotyledoside-bovine serum albumin conjugate was synthesized to immunize animals. The efficacy of the cotyledoside-conjugate in inducing an immunological response was ascertained in rabbits (n = 4) and sheep (n = 4) by determining cotyledoside antibody titres with an ELISA using cotyledoside-hen ovalbumin as antigen. The formation of anticotyledoside antibodies was induced in both rabbits and sheep following immunization with the cotyledoside-protein conjugate. Protection provided by the vaccine was demonstrated by challenging sheep (n = 4) with repeated, daily doses of cotyledoside (0.015 mg/kg) administered intravenously, commencing 45 days after the initial vaccination. One control animal died on Day 3 of the challenge period and the other was severely affected after administration of the third cotyledoside dose. The immunized ewes (n = 2) remained clinically unaffected and the challenge was suspended following six daily injections. Vaccination as a means of preventing krimpsiekte seems to be quite feasible and deserves further investigation.

Central role for the cardiotonic steroid marinobufagenin in the pathogenesis of experimental uremic cardiomyopathy.

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 microg/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.

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.

Involvement of marinobufagenin in a rat model of human preeclampsia.

BACKGROUND: Preeclampsia is a potentially devastating disorder of hypertension in pregnancy for which there is currently no definitive treatment short of delivery. The bufadienolide, marinobufagenin (MBG), an inhibitor of Na(+)/K(+) ATPase, has been found to be elevated in extracellular fluid volume-expanded hypertensive patients, a condition similar to preeclampsia. Thus, these studies sought to examine the role of MBG in our rat model of preeclampsia. METHODS AND RESULTS: Pregnant female rats were injected intraperitoneally with deoxycorticosterone acetate (DOCA) and given 0.9% saline as drinking water for the duration of their pregnancy. Urinary MBG was measured using a DELFIA immunoassay. Blood pressure was measured via the tail-cuff method. Injections of anti-MBG antibody were given intraperitoneally or intravenously to hypertensive pregnant rats. MBG was given intraperitoneally to pregnant rats. Uterine arterioles were dissected free and their diameters were measured before and after perfusion of MBG, ouabain, or digoxin. MBG was found to be elevated in the pregnant + DOCA + saline (PDS) rats compared to normal pregnant animals. In addition, when PDS rats were injected with anti-MBG antibody, there was a subsequent reduction in blood pressure. Administration of MBG in normal pregnant rats caused an elevation in blood pressure equivalent to the PDS model. Also, uterine vessel measurements showed an increased vasoconstrictive reactivity to MBG in the PDS animals vs. the normal pregnant controls; while no changes were observed with perfusion of digoxin or ouabain at the same concentration. CONCLUSION: These results suggest a relationship between MBG and a syndrome in rats resembling preeclampsia. Armed with these promising results, it would seem logical to further examine the role of MBG in human preeclampsia.

Salt loading induces redistribution of the plasmalemmal Na/K-ATPase in proximal tubule cells.

BACKGROUND: We have reported that digitalis-like substances (cardiotonic steroids), including marinobufagenin (MBG), induce endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 cells. The current report addresses the potential relevance of plasmalemmal Na/K-ATPase redistribution to in vivo salt handling. METHODS: Male Sprague-Dawley rats were given 1 week of a high salt (4.0% NaCl) or normal salt (0.4% NaCl) diet. Urinary sodium excretion, as well as MBG excretion, was monitored, and proximal tubules were isolated using a Percoll gradient method. Tubular (86)Rb uptake, Na/K-ATPase enzymatic activity, and Na/K-ATPase alpha1 subunit density were determined. RESULTS: The high salt diet increased urinary sodium (17.8 +/- 1.8 vs. 2.5 +/- 0.3 mEq/day, P < 0.01) and MBG excretion (104 +/- 12 vs. 26 +/- 4 pmol/day), and decreased proximal tubular (86)Rb uptake (0.44 +/- 0.07 vs. 1.00 +/- 0.10, P < 0.01) and Na/K-ATPase enzymatic activity (5.1 +/- 1.1 vs. 9.9 +/- 1.6 micromol/mg pr/hr, P < 0.01) relative to the normal diet. Proximal tubular Na/K-ATPase alpha1 protein density was decreased in the plasmalemma fraction but increased in both early and late endosomes following the high salt diet. In rats fed a high salt diet, anti-MBG antibody caused a 60% reduction in urinary sodium excretion, substantial increases in proximal tubule (86)Rb uptake, and Na/K-ATPase enzymatic activity, as well as significant decreases in the early and late endosomal Na/K-ATPase alpha1 protein content. CONCLUSION: These data suggest that redistribution of the proximal tubule Na/K-ATPase in response to endogenous cardiotonic steroids plays an important role in renal adaptation to salt loading.