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(Hypertension. 2001;37:462.)
© 2001 American Heart Association, Inc.

Scientific Contributions

Marinobufagenin, an Endogenous -1 Sodium Pump Ligand, in Hypertensive Dahl Salt-Sensitive Rats

Olga V. Fedorova; Nikolai I. Kolodkin; Natalia I. Agalakova; Edward G. Lakatta; Alexei Y. Bagrov

From the Laboratory of Cardiovascular Science, Intramural Research Program, National Institute on Aging, Baltimore, Md (O.V.F., N.I.A., E.G.L., A.Y.B.), and Institute of Highly Pure Biopreparations, St. Petersburg, Russia (N.I.K.).

Correspondence to Alexei Y. Bagrov, Laboratory of Cardiovascular Science, Intramural Research Program, National Institute on Aging, 5600 Nathan Shock Drive, Baltimore, MD 21224. E-mail bagrovA{at}grc.nia.nih.gov

	Abstract

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Dahl salt-sensitive rats (DS), which have a mutation in the -1 subunit of Na⁺/K⁺-ATPase, exhibit impaired pressure natriuresis and on a high-salt diet, retain Na⁺ and exhibit increased blood pressure. Recently, we have shown that mammalian tissues contain a bufadienolide Na⁺/K⁺-ATPase inhibitory factor, marinobufagenin (MBG), that exhibits greater affinity for the -1 than -3 sodium pump isoform. The present study investigated the possible role of MBG in hypertension in DS on a high NaCl intake. Eight DS and 8 Dahl salt-resistant rats (DR) were placed on an 8% NaCl diet. Within 2 weeks, systolic blood pressure increased in DS (162±9 mm Hg at week 2 versus 110±2 mm Hg in baseline, P<0.01), and increased less in DR (124±3 mm Hg at week 2 versus 112±2 mm Hg in baseline). Renal excretion of MBG increased 4-fold (38.9±7.6 pmol versus 9.1±1.3 pmol in baseline, P<0.01) in DS, but by only 25% in DR (13.2±0.9 pmol versus 10.3±0.7 pmol in baseline). Excretion of endogenous ouabain did not change in either strain. MBG-immunoreactive material was purified from the urine of hypertensive DS by means of 2 steps of reverse-phase high performance liquid chromatography (HPLC) and compared with plant ouabain and amphibian MBG for its ability to inhibit the Na⁺/K⁺-ATPase from rat kidney (which expresses only -1 Na⁺/K⁺-ATPase isoform). Unlike ouabain (IC₅₀=248 µmol/L), serially diluted, HPLC-purified MBG immunoreactivity from DS and authentic MBG potently inhibited rat kidney Na⁺/K⁺-ATPase (IC₅₀=70 and 78 nmol/L, respectively). Our results suggest that an -1 Na⁺/K⁺-ATPase ligand, MBG, is elaborated to promote natriuresis in hypertensive DS. MBG acts as a selective inhibitor of the ouabain-resistant -1 Na⁺/K⁺-ATPase subunit, ie, the major sodium pump isoform of the kidneys, as would be expected of a putative natriuretic hormone.

Key Words: NaCl • Dahl rats • Na⁺/K⁺-ATPase • kidney • bufadienolides • marinobufagenin

	Introduction

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The Dahl-deWardener-Blaustein concept of natriuretic hormone postulates that in volume-expanded forms of hypertension, an enhanced production of endogenous digitalis-like sodium pump ligand (SPL) occurs with a primarily adaptive aim, to decrease the volume of circulating fluid by means of inhibition of the Na⁺/K⁺-ATPase in renal tubules. The excessive SPL production also contributes to hypertension by means of inhibition of the Na⁺/K⁺-ATPase in cardiovascular tissues.^{1 2 3} Several SPL have been described in mammalian tissues, including a cardenolide, ouabain-like compound (OLC),^{4 5} and bufadienolides,^{6 7 8} including marinobufagenin (MBG) -immunoreactive factor.⁹ MBG (3ß,5ß-dihydroxy-14,15-epoxybufadienolide) was originally discovered in amphibia.¹⁰ Subsequently, MBG immunoreactive material purified from human urine exhibited mass-spectral properties identical to those of amphibian MBG.¹¹ In contrast to ouabain, MBG exhibits a greater affinity for the ouabain-resistant -1 subunit of Na⁺/K⁺-ATPase^{12 13} , the main sodium pump isoform in renal tubules. In vitro, MBG exhibits vasoconstrictor actions.^{14 15} Elevated concentrations of plasma MBG immunoreactivity have been documented in several volume expanded and hypertensive states.^{15 16 17 18}

Hypertension that develops in Dahl salt-sensitive rats (DS) on a high NaCl intake is associated with retention of sodium and fluid, resulting from impairment of renal pressure natriuresis mechanisms.^{1 19} One of the factors that determines the blunted pressure natriuresis in DS is a mutation of the -1 subunit of Na⁺/K⁺-ATPase.²⁰ The mutated renal sodium pump of DS exhibits an abnormal Na/K pumping ratio, which on a high NaCl intake, results in the inability of the kidney to fully excrete sodium.^{21 22} This scenario, ie, a sodium pump abnormality and fluid retention, would be expected to elicit the enhanced production of a putative natriuretic hormone.

Recently, we compared regulation of MBG and OLC by acute NaCl loading in DS and in Dahl salt-resistant rats (DR).¹³ In both DS and DR, plasma and urinary OLC exhibited transient, 2- to 3-fold increases within 1 hour of NaCl loading, followed by a return to baseline levels. Plasma and urinary MBG increased in both strains within 1 hour of NaCl loading and, in contrast to OLC, remained elevated. The 8-hour MBG excretion was 4-fold greater in DS than in DR. DS exhibited a smaller natriuretic response than DR, despite a greater plasma Na⁺. Thus, acute salt loading of DS causes a transient increase in OLC but sustained increases in MBG tissue levels and excretion. We interpreted these results to indicate that an increased MBG production occurred in an attempt to compensate for the impaired pressure natriuresis. The purposes of the present study were to compare renal excretion of MBG and OLC in DS and DR on a chronic high NaCl intake and to characterize MBG-immunoreactive material purified from urine of hypertensive DS.

	Methods

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General
The protocol of the study has been approved by the ACUC of the Gerontology Research Center (GRC), National Institute on Aging. Five-week-old DS (n=8) and DR (n=8) were obtained from Harlan Sprague-Dawley Inc, Indianapolis, IN. The experiments begun after an adaptation period of 1 week. All rats were maintained in a 26°C environment with a 12:12 hour light-dark cycle on a normal (0.5%) NaCl diet (ICN Biochemicals) and tap water ad libitum. During the subsequent 14-day experimental period, an 8% NaCl diet (ICN Biochemicals) and tap water ad libitum was administered. Body weight, systolic blood pressure (SBP), water consumption, and urine output were measured, and SBP was recorded by tail-cuff plethysmography (IITC Model 31 IITC Life Science). Urinary samples were stored for measurements of Na⁺, K⁺ (Beckman Instrument, Synchron, EL/ISE) and SPL. At 14 days, rats were anesthetized with 10 mg/kg ketamine and killed by exsanguination from abdominal aorta.

Immunoassays
The MBG and OLC were measured in C18 extracted urine and plasma and high performance liquid chromatography (HPLC) fractions by means of solid phase fluoroimmunoassay as described recently in detail.¹³ The cross-reactivity of MBG antibody is as follows(%): MBG, 100; ouabain, 0.1; digitoxin, 3.0; digoxin, bufalin, and cinobufagin, 1.0; prednisone, spironolactone, and progesterone, <0.1; proscillaridin, <1.0; mixture of bufadienolides from Bufo marinus venom except MBG, <5. The OLC assay was based on rabbit ouabain antibody (1:150 000, Chemicon International Inc). The cross-reactivity of ouabain antibody is (%) as follows: ouabain, 100; digitoxin, 7.4; progesterone, <0.01; 5-beta cholanic acid, prednisone, and canrenoic acid, <0.01; proscillaridin, 0.2; MBG-free mixture of bufadienolides from Bufo marinus toad venom, 0.26; bufalin, 0.03; aldosterone, 0.09; MBG, 0.5.

HPLC
The procedure of partial purification of SPL included repeated reverse-phase HPLC fractionation and separation of fractions with MBG immunoreactivity (MBG-ir) as reported previously.¹¹ Five liters of urine obtained from DS during week 2 of an 8% NaCl intake were extracted with chloroform and prepurified by means of thin-layer-chromatogrpahy.¹¹ The resultant material was fractionated on a reverse phase column (Dinamax 60 A C18 22x300, 30 minutes, 10 mL/min detection at 220 nm) in a linear gradient of acetonitrile (0 to 90%) using a Gilson HPLC system (Model 303, detector model 116). Sixty 5-mL fractions were collected and analyzed for MBG-ir. Fractions that demonstrated the highest levels of MBG-ir were combined, lyophilized, and submitted to a second HPLC fractionation (Dinamax 60 A C18 22x300, 30 minutes, 10 mL/min, detection at 220 nm) in a linear gradient of acetonitrile (25 to 45%). Sixty 5-mL fractions were collected and analyzed for MBG-ir. Those HPLC fractions that exhibited the highest levels of MBG-ir were combined, lyophilized, and tested for their ability to react with MBG or ouabain antibody, and to inhibit the Na⁺/K⁺-ATPase from rat kidney.

Na⁺/K⁺-ATPase Studies
Na⁺/K⁺-ATPase was partially purified from kidney outer medulla of eight 10-week-old male Wistar rats as described recently using method of Jorgensen²³ with several modifications reported recently in detail.¹³ Na⁺/K⁺-ATPase activity was measured as reported previously¹³ with minor modifications. To increase the permeability of sealed membrane vesicles, membranes were preincubated for 30 minutes at room temperature with alamethicin (0.5 g/1 g of protein). Aliquots of membrane suspensions (100 µL containing 1 µg protein/well) were preincubated for 60 minutes at 37°C with amphibian MBG, plant ouabain, or HPLC-purified MBG-immunoreactive material, and then incubated for 15 minutes at 37°C in 96-well plates in the assay medium (mmol/L): Na 100, K 5, MgCl₂ 6, EDTA 1, Tris 50, ATP 7, NaN₃ 5 (pH=7.4). The reaction was stopped by the addition of 0.1 mL of quenching solution (1 N sulfuric acid, 0.5% ammonium molybdate), followed by the color reaction with 0.02% SnCl₂. Total ATPase activity was measured by the production of inorganic phosphate (P_i), and Na⁺/K⁺-ATPase activity was estimated as the difference between total ATPase activity in the absence and in the presence of 5 mmol/L ouabain.

Statistics
Results are reported as means±SEM. Statistical analyses of the measured variables were assessed by 1-way ANOVA followed by Newman-Keuls test, 2-tailed t test (when appropriate), or nonlinear regression using GraphPad Prism software (GraphPad Inc). A P value less than 0.05 was considered significant.

	Results

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Figure 1 illustrates sodium intake and excretion, diuresis, systolic blood pressure, and SPL levels in DS and DR during 0.5% NaCl intake, and after 2 weeks of an 8% NaCl diet. During the lower NaCl intake, no differences between the 2 strains in the above parameters were detected. However, while on a high NaCl diet, DS exhibited less natriuresis than DR (Figure 1A) in the presence of equal NaCl intake (Figure 1B), but had comparable diuresis to that of DR (Figure 1C). After 2 weeks of a high NaCl, systolic blood pressure in DS increased by 42 mm Hg and renal excretion of MBG increased 4-fold, but excretion of OLC did not change from its baseline value. In contrast to DS, DR systolic blood pressure increased by 12 mm Hg after 2 weeks of a high NaCl intake, and MBG excretion rose by only 25%. As in DS, OLC excretion did not differ from baseline. Plasma MBG in DS after 2 weeks of a high NaCl administration was 3 times higher than in DR (1.30±0.27 nmol/l and 0.42±0.06 nmol/l, respectively, P<0.01). Conversely, plasma levels of OLC in DR exceeded those in DS by 2.5 times (0.33±0.05 and 0.12±0.01 nmol/l, P<0.01).

View larger version (40K): [in this window] [in a new window]   Figure 1. NaCl excretion and intake, diuresis, systolic blood pressure, and renal excretion of MBG and OLC in DS and DR on an 0.5% NaCl intake and after 2 weeks of administration of 8% NaCl diet. Means±SEM from 8 observations. *P<0.01, **P<0.001 versus 0.5% NaCl intake, #P<0.01 versus DR on an 8% NaCl intake. One-way ANOVA followed by Newman-Keuls test.

Figure 2A illustrates the absorbance profile and distribution of MBG-ir among 60 half-minute fractions eluting from reverse-phase HPLC columns. More than 90% of MBG-ir eluted in fractions 39 and 40. These were combined and further fractionated on a semipreparative reverse-phase column. Eighty percent of the total MBG-ir was eluted in fractions 48 to 50 (Figure 2B). The material from fractions 48 to 50 was lyophilized and tested for its ability to interact with the MBG or ouabain antibody and to inhibit the Na⁺/K⁺-ATPase from rat kidney. Serial dilutions of material eluted in HPLC fractions 48 to 50 paralleled the calibration curve of MBG (Figure 2C) but not that of ouabain (Figure 2D).

View larger version (47K): [in this window] [in a new window]   Figure 2. Purification of MBG-immunoreactive compound from urine of hypertensive DS. (A) Elution of MBG-immunoreactive material (bars) from reverse-phase Dinamax HPLC column in a 0 to 90% linear gradient of acetonitrile. Solid line, absorption at 220 nm. (B) Elution of MBG immunoreactive material (bars) from reverse-phase Dinamax HPLC column in a 25 to 45% linear gradient of acetonitrile. Solid line, absorption at 220 nm. (C) Inhibition of binding of MBG antiserum to immobilized MBG-RNAase conjugate by standards of amphibian MBG (•), serially diluted MBG immunoreactive material from DS () and ouabain (). (D) Inhibition of binding of ouabain antiserum to immobilized ouabain-ovalbumin conjugate by standards of amphibian MBG (•), serially diluted MBG immunoreactive material from DS () and ouabain (). (E) Inhibition of Na+/K+-ATPase from rat kidney by amphibian MBG, serially diluted MBG immunoreactive material from DS and ouabain. Each point represents means±SEM from 5 to 8 experiments.

Concentration-response curves of Na⁺/K⁺-ATPase inhibition by amphibian MBG, ouabain, and HPLC-purified MBG-ir from DS are presented in Figure 2E. Compared with ouabain (IC₅₀=248 µmol/l), MBG exhibited a greater inhibition of Na⁺/K⁺-ATPase (IC₅₀=78 nmol/l). MBG-immunoreactive material isolated from DS inhibited the renal Na⁺/K⁺-ATPase in a manner similar to that of toad MBG (IC₅₀=70 nmol/l). The curves of Na⁺/K⁺-ATPase inhibition were further analyzed using a 2 site competition model. The IC_50’s corresponding to the inhibition occurring at the level of high- and low-affinity sites are listed in Table 1.

View this table: [in this window] [in a new window]   Table 1. Interactions of MBG and Ouabain with Renal Na+/K+-ATPase

	Discussion

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The major findings of the present experiment are that (1) development of hypertension in DS on a chronic (2-week) high NaCl intake is associated with substantial increases in renal excretion of MBG, and (2) HPLC-purified MBG-immunoreactive material from DS potently inhibits ouabain-insensitive renal Na⁺/K⁺-ATPase.

Despite significantly elevated blood pressure, DS exhibited less natriuresis than DR, although sodium intake and diuresis were comparable in both strains. On a high NaCl intake, DS, due to altered Na⁺/K⁺ pumping ratio of the Na⁺/K⁺-ATPase, could exhibit deficient intestinal sodium absorption. However, the fact that previously no differences in intestinal Na⁺/K⁺-ATPase activity and kinetics in DS and DR on an 8% NaCl were reported²⁴ argues against such a possibility.

The enhanced production of an endogenous ligand of -1 Na⁺/K⁺-ATPase, such as MBG, seems to be an adaptation in response to the inability of DS to fully accommodate excessive sodium intake. The importance of this mutation can be illustrated by a recent experiment in which transgenic DS expressing the wild-type -1 isoform on a high-NaCl diet developed less of a blood pressure increase, and exhibited a lower mortality and less renal damage, compared with nontransgenic DS.²⁰

The experiments with cross-circulation by Dahl et al provided the first evidence that a circulating pressor substance is responsible for elevations of blood pressure in DS on a high NaCl intake.¹ Later, in a series of experiments, Leenen and coworkers demonstrated that a centrally acting "endogenous ouabain" plays an important role in the establishment of salt-induced hypertension in DS by means of the brain renin-angiotensin system.^{25 26 27 28} Our present results demonstrate that in DS, MBG, rather than OLC, is increased, within 2 weeks of administration of an 8% NaCl diet, suggesting that the former is more likely to contribute to the maintenance of elevated blood pressure. Our data are in keeping with the observation that, on a high NaCl intake, plasma OLC in DR, but not in DS, becomes elevated.²⁹ The importance of higher plasma levels of OLC in salt-loaded DR compared with DS, as well as the possible relationship between MBG and brain OLC, remains to be elucidated.

In the present study, the elution pattern of MBG-ir from reverse phase HPLC columns was similar to that observed in our previous work in which material from human plasma and urine was studied.^{9 11 15} MBG-ir eluted from the HPLC column with 42% acetonitrile, which is consistent with a relatively low polarity of MBG. When serially diluted MBG-immunoreactive material was studied for its ability to compete with immobilized ouabain and MBG conjugates for MBG and ouabain antibody in a solid phase fluoroimmunoassay, the displacement curve of MBG-ir paralleled that of MBG, but not of plant ouabain. When HPLC-purified MBG-immunoreactive material was analyzed for its ability to inhibit the Na⁺/K⁺-ATPase from rat kidney (Figure 2E), its inhibitory potency was similar to that of amphibian MBG. Both amphibian MBG and MBG-ir exhibited a greater inhibition of Na⁺/K⁺-ATPase in the kidney membranes, which occurred at a level of both high- and low-affinity binding sites. Conversely, plant ouabain-induced Na⁺/K⁺-ATPase inhibition occurred at a level of low-affinity binding only. In the nanomolar concentration range, both MBG-ir and amphibian MBG inhibited the renal Na⁺/K⁺-ATPase by 25%. Therefore, nanomolar concentrations of MBG observed in plasma of hypertensive DS are sufficient to induce a substantial inhibition of the renal sodium pump. Thus, in DS, development of a genetically determined, salt-sensitive hypertension is associated with increased renal excretion of a bufadienolide inhibitor of a ouabain-resistant sodium pump. We would suggest that the enhanced MBG production is a compensatory adjustment against the impaired pressure natriuresis.

	Acknowledgments

 
This study was undertaken while A.Y.B. held a National Research Council-NIA/NIH Senior Research Associateship. The authors are grateful to Laurie B. Reynolds and Karla M. Torres for excellent technical assistance.

Received October 25, 2000; first decision December 11, 2000; accepted December 19, 2000.

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