Monica Ianosi-Irimie

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.

A rat model of preeclampsia.

Preeclampsia/eclampsia is a disorder of human pregnancy that continues to exact significant maternal morbidity and mortality and fetal wastage. Therapy of these disorders has not changed in over 50 years and there are no proven preventive measures. We describe a model of the development of a syndrome in the pregnant rat that resembles preeclampsia, which results from the imposition of excessive volume expansion early in gestation. We administered desoxycorticosterone acetate (DOCA) to pregnant animals whose drinking water had been replaced with saline. We compared the results obtained in these animals with those resulting from the study of control, virgin animals, virgin animals receiving DOCA and saline, and normal pregnant (NP) animals. The virgin animals given DOCA and saline did not become hypertensive. The experimental paradigm in the DOCA plus saline pregnant (PDS) animals provides many of the phenotypic characteristics of the human disorder including the development of hypertension, proteinuria, and intrauterine growth restriction. In addition, the mean blood nitrite/nitrate concentration was reduced in the PDS rats compared with their NP counterparts. We propose that this model may prove to be useful in the study of the human condition.

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.

Beneficial Effects of Metolazone in a Rat Model of Preeclampsia

Preeclampsia is a disorder that continues to exact a significant toll with respect to maternal morbidity and mortality as well as fetal wastage. Furthermore, the treatment of this disorder has not changed significantly in 50 years and is unsatisfactory. The use of diuretics in this syndrome is controversial because there is a concern related to potential baleful effects of volume contraction leading to a possible further decrement in the perfusion of the maternal-fetal unit. Metolazone is a diuretic/antihypertensive agent, which has a therapeutic effect on blood pressure (BP) in human essential hypertension without causing a natriuresis. We administered the drug in nondiuretic doses in a rat model of preeclampsia previously developed in this laboratory. The drug reduced BP without an accompanying natriuresis. Although there was a trend toward an improvement in intrauterine growth restriction, as determined by litter size and the number of pups demonstrating malformations, the values did not reach statistical significance. We conclude that metolazone, in low dosage, is an effective antihypertensive in this rat model. These studies have implications for the treatment of the human disorder.

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.

414 ALTERED EXPRESSION OF RENAL NA/K ATPASE IN HYPERTENSIVE PREGNANT RATS TREATED WITH METOLAZONE

We have developed a model of preeclampsia in the rat that has several phenotypic characteristics of human preeclampsia, including hypertension, proteinuria and intrauterine growth restriction. Metolazone (M) is a thiazide-like diuretic/antihypertensive agent that can reduce blood pressure in hypertensive patients without causing a natriuresis. We administered the drug in dosages of 0.05 mg/kg BW to pregnant rats weighing 250-300 g rendered “preeclamptic” by the administration of desoxycorticosterone acetate and replacement of their drinking water with 0.9% saline. The preeclamptic (PDS) group attained mean tail cuff blood pressures (BP) of 128 ± 9 mm Hg at day 17 of pregnancy as compared to a mean value of 92 ± 14 mm Hg in a group given M (PDS + M) in a daily non-natriuretic dose by gastric gavage (p < .01). After normalization for β-actin, Western immunoblot determinations (triplicate) involving 7 PDS and 9 PDS + M animals showed a 36% decrease in the expression of Na/K ATPase in the kidney cortex from PDS + M compared to untreated PDS rats. A 32% decrease in expression in the renal medulla was also seen in the PDS + M animals compared to the PDS group. We conclude that the action of M in the reduction of BP in our “preeclamptic” rat model includes both its effects on Na transporters as well as the expression of the Na/K ATPase enzyme in the kidney.

209 ROLE OF NITRIC OXIDE IN THE PATHOGENESIS OF A PREECLAMPSIA-LIKE SYNDROME IN RATS

We have developed a model of preeclampsia in the rat by administering desoxycorticosterone acetate (DOCA) and replacing their drinking water with 0.9% saline. The preeclamptic group (PDS group) showed several phenotypic characteristics of human preeclampsia, including increased blood pressure (BP), intrauterine growth restriction (IUGR), and proteinuria, when compared to the normal pregnant (NP) control group. To better understand this syndrome, we measured the nitric oxide (NO) in blood. NO has been reported to be implicated in the pathogenesis of preeclampsia. We also studied two other pregnant animal groups, one receiving only saline but no DOCA (PS) and the second receiving only DOCA with normal drinking water (PD). The results are as follows (data are means ± SD): (Table) We conclude that DOCA is largely responsible for the IUGR but the complete preeclamptic syndrome will not develop without saline. A possible explanation would be that saline is producing an initial status of volume expansion with increased cardiac output. The latter may trigger exaggerated renal hypertrophy, activation of the RAA, as well as release of growth and vasoconstrictive (and natriuretic) factors. Similar observations have been made in the preeclamptic patient.

63 RESIBUFOGENIN REDUCES BLOOD PRESSURE IN A RAT MODEL OF PREECLAMPSIA

Preeclampsia is a disorder which occurs only in human pregnancy. It consists of the development of hypertension, proteinuria and, often, excessive edema after 20 weeks of gestation in patients with previously normal blood pressure. It is the second most frequent cause of fetal wastage and maternal morbidity and mortality. Progress in understanding the pathophysiology of this disorder has been hampered by a lack of suitable animal models, especially of the early events in its pathogenesis. We have developed a rat model which reproduces many of the phenotypic characteristics of the human syndrome, including hypertension, proteinuria and intrauterine growth restriction. We have determined that the excretion of a circulating inhibitor of Na/K ATPase, marinobufagenin (MBG), a cardiotonic bufadienolide which has vasoconstrictive properties, is elevated in these animals even before hypertension develops. Resibufogenin (RBG) is a structurally related analogue that differs from MBG only by the absence of the 5 β-hydroxyl-grouping. Furthermore, this compound has been determined to have no effect on angiogenesis. We therefore injected this material intraperitonealy daily in a dose of 15.3 μg/kg in five pregnant animals which had been rendered “preeclamptic” by administering desoxycorticosterone (DOCA) and replacing their drinking water with 0.9% saline. Systolic blood pressure (BP), measured with a tail cuff method, which rose to 132 ± 5 mm Hg from an initial mean value of 109 ± mm Hg (p<0.001) due to the saline and DOCA treatment, fell to 117 ± 8 mm Hg after two days (p<0.01). This reduction in BP persisted throughout the treatment reaching a mean value of 115 ± 10 mm Hg (p = 0.02) after seven days. These data indicate that RBG is effective in reducing BP in this rat model of human preeclampsia. We postulate that its mechanism of action is that of competitive inhibition of the effect of MBG to inhibit Na/K ATPase. Studies to evaluate this possibility are under way.