Differing effects of resibufagenin on cinobufatalin- versus marinobufagenin-induced preeclampsia in a rodent model.

OBJECTIVE: Previous studies from this laboratory have demonstrated that the bufodienolide, marinobufagenin, causes a syndrome in the pregnant rat that resembles human preeclampsia. Furthermore, marinobufagenin urinary excretion is elevated in approximately 85% of preeclamptic patients. Resibufagenin, an antagonist to marinobufagenin, completely prevents the syndrome (hypertension, proteinuria, and intrauterine growth restriction) if given from early pregnancy. STUDY DESIGN: We investigated the effects of another bufodienolide, cinobufatalin, to determine if it, likewise, could induce the rat "preeclamptic" syndrome, which it did. We then examined whether resibufagenin could prevent the syndrome due to cinobufatalin. RESULTS: Resibufagenin improved hypertension but not proteinuria, and did not prevent uterine growth restriction. CONCLUSION: We conclude that more than one bufodienolide may induce the preeclamptic syndrome and that each may require a specific antagonist to prevent (or treat) the syndrome.

Marinobufagenin regulates permeability and gene expression of brain endothelial cells.

Marinobufagenin (MBG) is a cardiotonic steroid that increases in the circulation in preeclampsia. Preeclampsia and eclampsia are associated with cerebral edema. Therefore, we examined the effects of MBG on human brain microvascular endothelial cells (HBMEC) in vitro. MBG enhanced the permeability of HBMEC monolayers at 1-, 10-, and 100-nM doses, but had no effect at 0.1 nM. Agilent Human Gene Expression microarrays were utilized in these studies. MBG treatment (10 nM for 12 h) downregulated concentrations of the soluble VEGFR transcript sFLT by 59% but did not alter those of FLTv3 mRNA (determined by quantitative PCR). When treated and control HBMEC transcriptomes were interrogated on microarrays, 1,069 genes appeared to be regulated by MBG. Quantitative RT-PCR confirmed that MBG treatment upregulated ENKUR mRNA concentrations by 57%. Its protein product interacts with calmodulin and calcium channel proteins. MBG treatment downregulated several genes whose protein products are involved in cell adhesion (ITGA2B, FERMT1, CLDN16, and TMEM207) and cell signaling (GRIN2C, SLC8A1, and ESR1). The level of downregulation ranged from 22 to 66%. Altogether, MBG actively enhanced the permeability of HBMEC monolayers while downregulating genes involved in adhesion. MBG treatment had variable effects on ENKUR, GRIN2C, and SLC8A1 genes, all associated with calcium transport. These studies provide the basis for future investigations of MBG actions in normal physiology and disease.

Marinobufagenin is an upstream modulator of Gadd45a stress signaling in preeclampsia.

Preeclampsia (PE) is a hypertensive disorder of pregnancy, in which marinobufagenin (MBG), a circulating cardiotonic steroid, is increased. The Gadd45a stress sensor protein is an upstream modulator of the pathophysiological changes observed in PE. However, the effects of MBG on Gadd45a stress signaling remain unknown. We examined the expression of Gadd45a, the sFlt-1 receptor, and p38, as well as caspase 3 and 8 activities in placental samples from four groups of rats. These were: normal pregnant (NP, n=8); pregnant rats which received weekly injections of desoxycorticosterone acetate and 0.9% saline as their drinking water (PDS, n=9); normal pregnant rats injected with MBG (NPM, n=8); and PDS rats injected with resibufogenin (RBG), an in vivo antagonist of MBG (PDSR, n=8). Utilizing human cytotrophoblast (CTB) cells, we examined the effect of MBG on these stress signaling proteins in vitro. Placental Gadd45a expression, caspase 3 and 8 activities, sFlt-1 concentrations, and sFlt-1 receptor expression were significantly higher in PDS and NPM compared to NP and PDSR rats. Gadd45a protein was significantly upregulated in the CTB cells when MBG was present in concentrations ≥1nM. Treatment with MBG (≥1nM) also significantly arrested cell cycle progression and activated the expression of the Gadd45a-mediated stress signaling proteins. Inhibition of Gadd45a through RNAi-mediation attenuated MBG-induced CTB cell stress signaling. In conclusion, MBG is involved in the alteration in Gadd45a stress signaling both in vivo and in vitro and RBG prevents these changes when administered in vivo.

Marinobufagenin predicts and resibufogenin prevents preeclampsia: a review of the evidence.

OBJECTIVE: The purpose of this review is to provide information detailing the existing evidence with regard to the hypothesis that marinobufagenin (MBG) is an important etiologic and predictive factor in preeclampsia (PE). In addition, evidence describing the role of the antagonist to MBG, resibufogenin (RBG), in the prevention and/or treatment of this disorder is provided. STUDY DESIGN: The studies outlined were performed in an animal model of PE, in in vitro experiments, and in human studies. RESULTS: Data have been obtained that strongly support the hypothesis that ~60 to 70% of PE patients demonstrate elevations in urinary and serum MBG levels. In the animal model, the entire syndrome can be prevented by the administration of RBG beginning early in pregnancy. CONCLUSION: Expanded human trials of MBG as a predictor of the later development of PE are warranted as are studies of the efficacy and safety of RBG as a preventative/therapy.

Marinobufagenin levels in preeclamptic patients: a preliminary report.

Preeclampsia is a disorder resulting in significant fetomaternal complications with no definitive pharmacological intervention. A bufadienolide, marinobufagenin, has been implicated in the etiology of preeclampsia. We investigated both the blood and urine levels of marinobufagenin in preeclamptic and control subjects. Preeclamptic and normotensive pregnant women were recruited at various gestational age periods. Blood and urine specimens were obtained and analyzed for marinobufagenin levels and creatinine. The former determination was performed utilizing a new, novel chemifluorescent enzyme-linked immunosorbent assay. The marinobufagenin levels were higher in preeclamptics than in the controls in both serum and urine at various gestational age periods. Additionally, the mean level of marinobufagenin in the preeclamptic group was significantly greater than in controls in both blood and urine specimens ( P < 0.05). These data are consistent with a role for marinobufagenin in the etiology of preeclampsia. This study demonstrated comparable results in blood and urine samples. This suggests that subsequent studies on levels of marinobufagenin as a screening test for preeclampsia could be done utilizing urine samples, which are easier to obtain, less invasive, more cost-effective, and as accurate as the serological tests.

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.

Emerging role of the bufadienolides in cardiovascular and kidney diseases.

The bufadienolides are a group of steroid hormones that circulate in blood and are excreted in urine. They have the ability to inhibit the adenosine triphosphatase sodium-potassium pump (Na(+)-K(+)-ATPase), with predilection for its alpha1 isoform. This capability enables them to share with other cardiac glycosides the facility to cause an increase in sodium excretion, produce vasoconstriction resulting in hypertension, and act as cardiac inotropes. Bufadienolides have been implicated in instances of volume expansion-mediated hypertension, syndromes in which they are considered capable of causing a vascular leak, interfering with cellular proliferation, and inhibiting cellular maturation. An antagonist to the most well-studied bufadienolide, marinobufagenin, is resibufogenin, a compound that provides promise for the treatment of disorders in which excessive levels of marinobufagenin are present and are etiopathogenetic.

Alterations in the renin-angiotensin system in a rat model of human preeclampsia.

BACKGROUND/AIMS: Preeclampsia is a hypertensive disorder unique to pregnancy in which elevated levels of marinobufagenin (MBG) have been reported. The renin-angiotensin system (RAS) may also play a role in the pathogenesis of preeclampsia. The aim of our study was to evaluate the status of the RAS in a rat model of preeclampsia characterized by hypertension, proteinuria, excessive weight gain and intrauterine growth restriction. METHODS: We evaluated the components of the RAS in 5 groups of animals: nonpregnant control; normal pregnant (NP); pregnant rats which received injections of desoxycorticosterone acetate and 0.9% saline as their drinking water (PDS); normal pregnant rats injected with MBG (NPM), and PDS rats to which resibufogenin (RBG) had been administered (PDSR). RBG is an antagonist of MBG differing in structure from MBG only in the absence of a hydroxyl group in the beta-5 position. RESULTS: Plasma levels of active renin, renin and Ang II were significantly lower in PDS and NPM compared to NP and PDSR rats (p < 0.05). However, placental levels of these components were increased significantly in PDS and NPM compared to NP and PDSR rats (p < 0.05). Placental AT(1) receptor expression was significantly higher in PDS and NPM compared to NP and PDSR rats (p < 0.05). CONCLUSIONS: (1) The peripheral RAS is downregulated, and the uteroplacental RAS is upregulated in this rat model of preeclampsia; (2) MBG is involved in the causation of these alterations, and (3) RBG prevents these changes.

Genetic variation in solute carrier genes is associated with preeclampsia.

OBJECTIVE: The objective of the study was to evaluate allelic variation in 26 members of the solute carrier (SLC) gene family for an association with preeclampsia. STUDY DESIGN: Preeclampsia cases were women with mild or severe preeclampsia. Controls were enrolled from women without hypertension-related complications who presented for delivery at term (≥37 weeks). The association between preeclampsia and SLC gene single-nucleotide polymorphisms (SNPs) and haplotypes was evaluated by logistic regression models. RESULTS: Rs4957061 in SLC9A3 was significantly associated with a reduced risk of preeclampsia in whites (T allele, P = .002; odds ratio [OR], 0.33; 95% confidence interval [CI], 0.16-0.67). For SLC4A1 in blacks, the G allele of rs2074107 (P = .004; OR, 1.56; 95% CI, 1.15-2.12) and the A allele of rs2857078 (P < .001; OR, 1.67; 95% CI, 1.27-2.17) were significantly associated with preeclampsia. Also in blacks, rs10066650 in SLCO4C1 (G allele, P = .002; OR, 1.72; 95% CI, 1.21-2.46) was significantly associated with increased risk. Sliding window haplotype analyses identified significantly associated haplotypes in these genes. CONCLUSION: SNPs and haplotypes in SLC9A3 in whites and SLC4A1 and SLCO4C1 in blacks are significantly associated with preeclampsia.

Vascular leak in a rat model of preeclampsia.

BACKGROUND/AIMS: Preeclampsia is a hypertensive disorder which develops de novo in women during pregnancy. The urinary excretion of the cardiotonic steroid, marinobufagenin (MBG), is increased prior to the development of hypertension. Preeclamptic patients are volume expanded but much of the excess salt and water appears to be located primarily in the interstitial space. Therefore, 'capillary leak' syndrome has been postulated in this disorder. METHODS: We evaluated the vascular leakage in normal rats following MBG injection and in a rat model of human preeclampsia. We measured the changes in light intensity comparing that in the intravascular to the extravascular space by assessing 'leak' of fluorescein-labeled albumin (FITC-albumin) from mesenteric postcapillary venules. RESULTS: FITC-albumin extravasation continued to increase in a time-dependent fashion after MBG infusion and was significant (p < 0.05) at 60 min of observation when compared to sham rats. We also observed a significant difference in 'vascular leakage' in preeclamptic rats compared to control non-pregnant and normal pregnant groups starting at 20 min after the FITC-albumin infusion. CONCLUSION: We propose that MBG is involved in the production of a 'vascular leak' in our rat model of preeclampsia.

Vascular effects of the bufodienolides.

The bufodienolides are natriuretic steroids, which also have the capacity to cause vasoconstriction, and are cardiac inotropes. Their mechanism of action appears to be related to their ability to inhibit Na+/K+ ATPase. The actions of one of these compounds, marinobufagenin (MBG), have been investigated in a rat model of preeclampsia, an example of volume expansion-mediated hypertension. The urinary excretion of MGB is increased in this model. Furthermore, this increment in its excretion occurs prior to the development of hypertension and proteinuria. The animals also demonstrate intrauterine growth restriction. Studies of the effect of MBG on cytotrophoblast cells reveal that MGB inhibits the migration, proliferation and invasion of these cells. We propose that MGB is an important etiologic factor in at least some forms of preeclampsia and that the level of its excretion in the urine may prove to be of diagnostic value.

Uncoupled endothelial nitric oxide synthase and oxidative stress in a rat model of pregnancy-induced hypertension.

BACKGROUND: Preeclampsia is a human pregnancy-associated syndrome associated with hypertension, proteinuria, and endothelial dysfunction. We tested whether increased reactive oxygen species (superoxide and peroxynitrite) production and decreased bioavailability of the endothelial nitric oxide (NO) synthase (eNOS) cofactor tetrahydrobiopterin (BH4) contributes to maternal endothelial dysfunction in rats with pregnancy-induced hypertension and several characteristics of preeclampsia. METHODS: Nonpregnant (DS) and pregnant (PDS) rats were treated with deoxycorticosterone acetate and 0.9% saline for approximately 3 weeks and nonpregnant (Con) and pregnant (P) rats received tap water. Blood pressure, urinary protein levels, mesenteric vascular reactivity, aortic protein expression, and aortic reactive oxygen species levels were compared between the four groups. RESULTS: The PDS rats had significantly decreased mesenteric endothelium-dependent relaxation responses and aortic NO production compared to Con, DS, and P rats despite increased aortic eNOS expression. Aortic superoxide and peroxynitrite levels were increased in PDS rats compared with Con, DS, and P rats. Scavenging of reactive oxygen species or increasing tetrahydrobiopterin levels normalized mesenteric endothelium-dependent relaxation responses, aortic NO production, and aortic superoxide and peroxynitrite levels in PDS rats. CONCLUSIONS: These data suggest that increased superoxide production by NADPH oxidase, peroxynitrite degradation of BH4, and uncoupled eNOS contribute to endothelial dysfunction in a rat model of pregnancy-induced hypertension.

Phenotypic characteristics shared by preeclamptic patients and an animal model of the syndrome: report of a pilot study.

BACKGROUND: Preeclampsia is a disorder that affects between 3% and 10% of all pregnancies. Progress in the understanding of the etiology (or etiologies) of this disorder has been impeded by the lack of suitable animal models of its early pathogenesis. Etiologic possibilities abound, and there are a number of considerations that suggest that preeclampsia is not one disease but rather a group of diseases with similar phenotypic characteristics. A rat model of this syndrome has been developed by inducing excessive volume expansion using desoxycorticosterone acetate and by replacing the drinking water with 0.9% saline. These animals develop hypertension, proteinuria, and intrauterine growth restriction (IUGR). However, they do not develop glomerular endotheliosis or a reduced glomerular filtration rate (GFR). We therefore surveyed the charts of patients with a discharge diagnosis of preeclampsia. We addressed the question of whether there was a group of such patients with the characteristics of our rat model. These include hypertension, proteinuria, IUGR, and either normal or only mildly abnormal GFR. METHODS: We performed a retrospective chart review of 630 consecutive patients discharged with a diagnosis of preeclampsia. Of the patients, 1290 had all data available to allow appropriate analysis. RESULTS: A total of 29 patients demonstrated hypertension (>140/90 mm Hg), proteinuria (>300 mg/ 24 h), and IUGR and did not have any confounding comorbid conditions. Of these 29 patients, 18 had GFR that were within the range expected for gestational age or only slightly reduced. CONCLUSIONS: There is a group of patients that mirror the characteristics of our animal model. Accordingly, at least one etiology of preeclampsia is related to excessive expansion of the extracellular fluid volume.

Resibufogenin corrects hypertension in a rat model of human preeclampsia.

The study of the pathogenesis of preeclampsia has been hampered by a relative dearth of animal models. We developed a rat model of preeclampsia in which the excretion of a circulating inhibitor of Na/K ATPase, marinobufagenin (MBG), is elevated. These animals develop hypertension, proteinuria, and intrauterine growth restriction. The administration of a congener of MBG, resibufogenin (RBG), reduces blood pressure to normal in these animals, as is the case when given to pregnant animals rendered hypertensive by the administration of MBG. Studies of Na/K ATPase inhibition by MBG and RBG reveal that these agents are equally effective as inhibitors of the enzyme.

Comparison of Neurocognitive Testing and the Measurement of Marinobufagenin in Mild Traumatic Brain Injury: A Preliminary Report.

The evaluation of concussed athletes, including testing to determine if and when they may return to play, has become an important task of athletic trainers and team physicians. Currently, concussion protocols are in place, which depend largely upon assessments based upon neurocognitive testing (NCT). The authors have evaluated the use of a biomarker of brain trauma, marinobufagenin (MBG), and compared its application in concussed athletes with the performance of NTC. We found a disparity between these two testing procedures. In this communication, the findings of these comparative data are presented. We noted that athletes whose NCT evaluations had returned to baseline and who were allowed to again participate in play then showed a recurrence of elevated urinary MBG excretion. These observations raise concern as to the processes currently in effect with regard to the decision as to returning athletes to the full activity. They suggest a need for further evaluation.

Preeclampsia. Part 1: clinical and pathophysiologic considerations.

Preeclampsia-eclampsia is still one of the leading causes of maternal and fetal morbidity and mortality. Despite active research for many years, the etiology of this disorder exclusive to human pregnancy is an enigma. Recent evidence suggests there may be several underlying causes or predispositions leading to the signs of hypertension, proteinuria, and edema, findings that allow us to make the diagnosis of the "syndrome" of preeclampsia. Despite improved prenatal care, severe preeclampsia and eclampsia still occur. Although understanding of the pathophysiology of these disorders has improved, treatment has not changed significantly in over 50 years. Although postponement of delivery in selected women with severe preeclampsia improves fetal outcome to a degree, this is not done without risk to the mother. In the United States, magnesium sulfate and hydralazine are the most commonly used medications for seizure prophylaxis and hypertension in the intrapartum period. The search for the underlying cause of this disorder and for a clinical marker to predict those women who will develop preeclampsia-eclampsia is ongoing, with its prevention the ultimate goal. This review begins with the clinical and pathophysiologic aspects of preeclampsia-eclampsia (Part 1). In Part 2, the experimental observations, the search for predictive factors, and the genetics of this disorder will be reviewed.

Preeclampsia. Part 2: experimental and genetic considerations.

Preeclampsia-eclampsia is still one of the leading causes of maternal and fetal morbidity and mortality. Despite active research for many years, the etiology of this disorder exclusive to human pregnancy is an enigma. Recent evidence suggests there may be several underlying causes or predispositions leading to the signs of hypertension, proteinuria, and edema, findings that allow us to make the diagnosis of the "syndrome" of preeclampsia. Despite improved prenatal care, severe preeclampsia and eclampsia still occur. Although understanding of the pathophysiology of these disorders has improved, treatment has not changed significantly in over 50 years. Although postponement of delivery in selected women with severe preeclampsia improves fetal outcome to a degree, this is not done without risk to the mother. In the United States, magnesium sulfate and hydralazine are the most commonly used medications for seizure prophylaxis and hypertension in the intrapartum period. The search for the underlying cause of this disorder and for a clinical marker to predict those women who will develop preeclampsia-eclampsia is ongoing, with its prevention the ultimate goal. This review began with the clinical and pathophysiologic aspects of preeclampsia-eclampsia (Part 1). Now, in Part 2, the experimental observations, the search for predictive factors, and the genetics of this disorder are reviewed.

Molecular effects of volume expansion on the renal sodium phosphate cotransporter.

BACKGROUND: Volume Expansion (VE) results in both natriuresis and a phosphaturia. In previous studies, Sprague-Dawley rats were infused with a modified saline solution. The expansion procedure resulted in a 70% increase in the phosphorylation of a 72 kDa proximal tubular brush border membrane (BBM) protein. In recent experiments, Sprague-Dawley rats were subjected to the same short term VE. For both control and VE animals, brush border membrane vesicles (BBMV) were obtained. METHODS AND RESULTS: Mass spectrometry of 3 proteins in the size range of our phosphoprotein resulted in the identification of ezrin/villin2, moesin, and PDZ domain-containing 1 (PDZ-dc1). Diphor-1 (currently renamed PDZ-dc1) is involved in regulation of the type II Na/Pi cotransporter. Ezrin and moesin are membrane-cytoskeletal linking proteins that are involved in the regulation of the sodium-hydrogen exchanger (NHE3) via interactions with another PDZ protein identified as sodium-hydrogen exchanger regulatory factor (EBP50, NHERF). Ezrin, moesin, and PDZ-dc1 protein levels were not increased following short term VE. Two-dimensional electrophoresis of our phosphorylated BBM proteins, followed by MALDI/MS analysis resulted in the identification of a protein mixture containing ezrin/moesin, alkaline phosphatase, and an unknown protein. Based on Western and immunoprecipitation data for ezrin, moesin, and PDZ-dc1 we believe that it is unlikely that our phosphoprotein is any of these 3 proteins. Parallels between NHE3 regulation (through EBP50/ERM proteins) and Na/Pi cotransporter regulation (through PDZ-dc1/ERM proteins) may be drawn. CONCLUSION: These changes in proximal Na/Pi cotransport may involve a signal transduction cascade including PDZ-dc1, ezrin, moesin, our phosphoprotein, and possibly other proteins.