The Na+K+-ATPase Inhibitor Marinobufagenin and Early Cardiovascular Risk in Humans: a Review of Recent Evidence.

PURPOSE OF REVIEW: This review synthesizes recent findings in humans pertaining to the relationships between marinobufagenin (MBG), a steroidal Na+/K+-ATPase inhibitor and salt-sensitivity biomarker, and early cardiovascular risk markers. RECENT FINDINGS: Twenty-four-hour urinary MBG strongly associates with habitual salt intake in young healthy adults (aged 20-30 years). Furthermore, in young healthy adults free of detected cardiovascular disease, MBG associates with increased large artery stiffness and left ventricular mass independent of blood pressure. These findings in human studies corroborate mechanistic data from rat studies whereby stimulation of MBG by a high salt intake or MBG infusion increased vascular fibrosis and cardiac hypertrophy. Twenty-four-hour urinary MBG may be a potential biomarker of early cardiovascular risk. Adverse associations between MBG-which increases with salt consumption-and early cardiovascular risk markers support the global efforts to reduce population-wide salt intake in an effort to prevent and control the burden of non-communicable diseases.

Functional and morphological aspects of thallium-induced nephrotoxicity in rats.

Until now the effect of thallium (Tl) on renal function has not been investigated systematically. Therefore, the dose (5, 10, 15, 20 mg Tl2SO4/kg body wt., intraperitoneally) and time-dependence of renal damage was investigated in diuresis experiments on conscious rats. Morphology was evaluated after perfusion fixation in situ. Morphologic changes were localized in the thick ascending limb of the loop of Henle, mostly expressed at the 2nd day after Tl administration, which were completely normalized again at the 10th day. Other parameters such as Tl concentration, changes in water content and the activity of Na+/K(+)-ATPase as well as the diuretic effect of furosemide confirmed the Tl effect to be localized in the renal medulla. One single Tl administration is followed by a decrease in glomerular filtration rate (GFR) and urine volume and an increase of proteinuria. Electrolyte excretion was only slightly changed. All changes were reversible within the 10-day investigation period.

A novel endogenous sodium-pump inhibitor in pig urine: purification and comparison with the inhibitor purified from bovine adrenal glands.

To clarify the chemical nature and pathophysiological significance of an endogenous, specific Na-pump inhibitor, extracts of various tissues have been examined. The activities were extracted by an Amberlite XAD-2 adsorbent and fractionated by high-performance liquid chromatography (HPLC) on an ODS column. Among many tissues and fractions tested, the extract of pig urine has been found to contain a large amount of 86Rb uptake inhibitory activities. The strongest inhibitory activity for 86Rb influx into erythrocytes also exhibited a cross-reactivity with anti-ouabain antibodies. This activity has been purified to homogeneity using five steps of reverse-phase HPLC. Although most of the dose-response curves for this purified Na-pump inhibitor, designated as uroxin, in the various assay systems paralleled those of ouabain and the inhibitor purified from bovine adrenal glands (designated as adrexin C), the cross-reactivity curve with anti-ouabain antibodies did not. The retention time of uroxin on an ODS HPLC column was also different from that of digoxin, ouabain, or adrexin C. 1H nuclear magnetic resonance spectroscopic study suggests that uroxin is a novel Na-pump inhibitor that is structurally different from any of the known cardiotonic steroids or the substances previously reported to exhibit Na-pump inhibitory activity. Thus, uroxin may be a new type of endogenous regulator for the Na pump.

Urinary excretion of digoxin-like immunoreactive factor and arginine-vasopressin in hyper- and hypo-thyroid rats.

1. Urinary excretion of digoxin-like immunoreactive factor and arginine-vasopressin and other parameters related to salt and water metabolism were studied in hyper- and hypo-thyroid rats after different tests. 2. Urinary excretion of arginine-vasopressin was increased in hyperthyroid and reduced in hypothyroid rats with respect to controls, in response to water deprivation or a hypertonic saline load. 3. Control and hypothyroid rats showed the highest urinary excretion of digoxin-like immunoreactive factor after a hypertonic saline load. However, hyperthyroid rats had the highest urinary levels of digoxin-like immunoreactive factor under normal conditions. 4. From these results it is suggested that: (a) hyper- and hypo-thyroid rats exhibit hyper- and hypo-responsiveness of arginine-vasopressin secretion to osmotic stimuli, respectively; (b) an unidentified digoxin-like immunoreactive factor measured in unextracted rat urine may be related to diuresis and natriuresis in control and hypothyroid rats; however, dissociation between this factor and natriuresis is observed in hyperthyroid rats.

Plasma atrial natriuretic factor and urinary excretion of a ouabain displacing factor and dopamine in normotensive pregnant women before and after delivery.

Estimation of urinary excretion of a ouabain displacing factor and dopamine was carried out immediately before delivery, and 7 days and 70 to 90 days after delivery in 12 normotensive pregnant women. Simultaneous estimation of plasma 99-126 atrial natriuretic factor, plasma renin activity, and plasma aldosterone were also undertaken. The data were compared with those obtained in a group of nonpregnant normotensive women (n = 14) and a group of pregnant normotensive women in the early phase of the third trimester (n = 14). Urinary ouabain displacing factor and dopamine levels were significantly higher in the early phase of the third trimester, as compared with nonpregnant women. But immediately before delivery, ouabain displacing factor excretion had fallen below nonpregnant values and dopamine excretion had dropped to control values. Both remained low after delivery. Plasma atrial natriuretic factor was higher in pregnant women, as compared with nonpregnant controls and remained high just before delivery and 7 and 70 to 90 days after delivery. Plasma renin activity and plasma aldosterone levels were higher during pregnancy and had fallen to nonpregnant values 7 days post partum. This drop in plasma renin activity and aldosterone by 7 days post partum, in contrast with the unchanged high values of atrial natriuretic factor, may contribute to negative sodium balance after delivery. It is concluded that there is considerable discrepancy in natriuretic and antinatriuretic factors before and after delivery.

The possible role of endogenous digitalis-like substance in the regulation of circadian changes in urinary electrolyte excretion in man.

The urinary volume (U.V.), Na excretion (UNaV) and K excretion (UKV) have been reported to show a circadian rhythm in man, but the mechanism of this rhythm has not been made clear. To investigate how atrial natriuretic peptide (ANP) and endogenous digitalis-like substance (DLS) participate in the circadian change in urinary electrolyte, the circadian changes in ANP and DLS (digoxin-like immunoactivity: DLI, Na-K-ATPase inhibitor: ATPI, ouabain binding inhibitor to Na-K-ATPase: OBI) were evaluated in 5 normal man. ANP, DLI and OBI showed no significant correlation with urinary electrolyte excretion, but there was a significant positive correlation between plasma ATPI and urinary Na excretion. From these results it is suggested that circulating Na-K-ATPase inhibitor (plasma ATPI) may be involved in the regulation of the circadian rhythm of urinary Na excretion.

[Sequential study of the plasmatic atrial natriuretic factor and the urinary excretion of an ouabain displacing factor and of dopamine in normotensive pregnant women before and after labor]. / Etude séquentielle du facteur atrial natriurétique plasmatique et de l'excrétion urinaire d'un facteur déplaçant l'ouabaïne et de la dopamine chez la femme enceinte normotendue avant et après l'accouchement.

Estimation of urinary excretion of a ouabain displacing factor (ODF) and dopamine was carried out immediately before delivery, 7 days and 70-90 days after delivery in 12 normotensive pregnant women. Simultaneous estimation of plasma 99-126 atrial natriuretic factor (ANF), plasma renin activity (PRA) and plasma aldosterone were also undertaken. The data were compared with those obtained in a non pregnant normotensive group of women (n = 14) and a group of pregnant normotensive women in the early third trimester (n = 14). Urinary ODF and dopamine were significantly higher in the early third trimester when compared with non pregnant women but immediately before delivery, ODF excretion had fallen below non pregnant values and dopamine excretion had dropped to control values. Both remained low after delivery. Plasma ANF was higher in pregnant women when compared with non pregnant controls and remained high just before delivery and 7 and 70-90 days after delivery. PRA and plasma aldosterone were higher during pregnancy and had fallen to non pregnant values 7 days post-partum. It is concluded that there is considerable discrepancy in the evolution of natriuretic and antinatriuretic factors before and after delivery and that the drop of PRA and aldosterone by 7 days post-partum, contrasting with the unchanged high values of ANF, may contribute to negative sodium balance after delivery.

Biochemical characterization of a digitalis-like substance in human urine, partially purified by high pressure liquid chromatography.

We have partially purified a digitalis-like extract from human urine by means of high pressure liquid chromatography (HPLC). Four fractions, giving positive activity for the cross-reaction in the digoxin radioimmunoassay, Na-K-ATPase inhibition or competition with ouabain binding to its receptor, were detected on HPLC. Among these fractions, the first (F-1) was shown to contain the principle fraction, sharing all the activities, and to exhibit quite similar characteristics to ouabain in the modes of Na-K-ATPase inhibition, ouabain displacement activity and positive inotropic action.

Biological activity of partially purified digitalis-like substance and Na-K-ATPase inhibitor in rats.

In order to study the biological activity of endogenous digitalis-like substance (DLS) and Na-K-ATPase inhibitor (ATPI), human urine was partially purified and administered to rats, and its effects on the urinary volume, urinary Na excretion and blood pressure (BP) were determined. In addition, the effect on myocardial Na-K-ATPase activity was also measured. After the extraction of 40L of urine with a reversed phase cartridge column (S-fraction), 20 ml of chloroform was added and extraction was repeated. The chloroform layer was applied to an open silica gel column, and at a fraction with ethylacetate: methanol (60: 40, T-1 fraction), DLS and ATPI were eluted at the highest concentration. The water layer was treated with charcoal (D-1 fraction). The acute administration of K-1, T-1 fraction to rats in vivo caused significant rises in urinary volume, urinary Na excretion and BP. In chronic administration of K-1 fraction, urinary Na excretion was significantly elevated and myocardial Na-K-ATPase activity was also significantly suppressed. These results suggest that DLS and ATPI cause increase in the urinary volume and urinary Na excretion and also possess a hypertensive action; and moreover, these substance may affect the heart like cardiotonic steroids and regulate BP by increasing cardiac contractility.

A ouabain-displacing factor in normal pregnancy, pregnancy-induced hypertension and pre-eclampsia.

1. A ouabain-displacing factor (ODF) was measured in the urine of non-pregnant, normotensive pregnant and hypertensive pregnant women by a receptor-binding assay with sodium, potassium-dependent adenosine triphosphatase. 2. Urinary ODF was significantly increased in normal pregnancy. 3. Greater increases were seen in pregnancy-induced hypertension and pre-eclampsia.

Endogenous Na+ transport inhibitor in human hypertension: further biochemical and chemical studies.

Endogenous digitalis-like compound(s) (endalin) has(ve) been reported to be involved in some diseases. Endalin activity is increased in plasma and urine of some essential hypertensives, and in Na+-dependent experimental hypertension. The aims of this study are to compare the biological properties of one endalin extracted from urine of hypertensive patients and of normotensive offspring of hypertensive subjects to those of ouabain and to determine the chemical nature of such an urine-derived endalin. The donors were selected on the basis of the highest Na+,K+-ATPase inhibition produced by extract from their 24-h urine. They consisted of 8 hypertensive patients, 21 normotensive subjects with family history of hypertension and 6 normotensive subjects with no known family history of hypertension. Endalin was semi-purified from 500 liters of pooled urine by flash chromatography on RP 18 packing (40 microns) followed by anion exchange chromatography and two HPLCs on RP 18 reversed phase. Endalin was traced by its capability of inhibiting dog kidney Na+,K+-ATPase activity and 3H-ouabain binding to the enzyme, by its cross-reaction with anti-digoxin antibodies and by its natriuretic effect in rat bioassay. The mechanism of Na+,K+-ATPase inhibition by a semi-purified urine-derived endalin and its consequences on Na-transport were studied and compared to those of ouabain. Semi-purified urine-derived endalin was similar to ouabain in that: it reversibly and specifically inhibited Na+,K+-ATPase activity; it inhibited Na+,K+-ATPase non-competitively with ATP; its inhibitory effect was facilitated by Na+; K+ decreased its inhibitory effect on Na+,K+-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Further biochemical characterization of an Na+ pump inhibitor purified from human urine.

An increase in endogenous Na+,K+-ATPase inhibitor(s) with digitalis-like properties has been reported in chronic renal insufficiency, in Na+-dependent experimental hypertension and in some essential hypertensive patients. The present study specifies some properties and some biochemical characteristics of a semipurified compound from human urine having digitalis-like properties. The urine-derived inhibitor (endalin) inhibits Na+,K+-ATPase activity and [3H]-ouabain binding, and cross-reacts with anti-digoxin antibodies. The inhibitory effect on ATPases of endalin is higher on Na+,K+-ATPase than on Mg2+-ATPase and Ca2+-ATPase. The mechanism of endalin action on highly purified Na+,K+-ATPase was compared to that of ouabain and was similar in that it reversibly inhibited Na+,K+-ATPase activity; it inhibited Na+,K+-ATPase non-competitively with ATP; its inhibitory effect was facilitated by Na+; K+ decreased its inhibitory effect on Na+,K+-ATPase; it competitively inhibited ouabain binding to the enzyme; its binding was maximal in the presence of Mg2+ and Pi; it decreased the Na+ pump activity in human erythrocytes; it reduced serotonin uptake by human platelets; and it was diuretic and natriuretic in rat bioassay. The endalin differed from ouabain in only three aspects: its inhibitory effect was not really specific for Na+,K+-ATPase; its binding to the enzyme was undetectable in the presence of Mg2+ and ATP; it was not kaliuretic in rat bioassay. Endalin is a reversible and partial specific inhibitor of Na+,K+-ATPase, its Na+,K+-ATPase inhibition closely resembles that of ouabain and it could be considered as one of the natriuretic hormones.

Sensitive assay and kinetic property of urinary inhibitor of Na+, K+-ATPase in sodium-loaded patients with essential hypertension.

A sensitive assay method to evaluate the inhibitor of Na+, K+-ATPase in human urine was developed by measuring the inorganic phosphate liberated from ATP in vitro using Na+, K+-ATPase from porcine cerebral cortex. Ouabain inhibited the Na+, K+-ATPase by competing with the potassium ion (an apparent Ki = 2.6 +/- 0.89 X 10(-8) M, n = 8) under the condition of 100 mM NaCl, 4.5 mM MgSO4 and 0.56 mM ATP. The apparent Km value of KCl was 0.4 mM. Factors inhibiting Na+, K+-ATPase were detected in the post-salt fraction on Sephadex G-15 chromatography following the ethanol extraction of lyophilized fresh urine of sodium loaded human subjects (300 meq Na+/day, for 4 days) with essential hypertension. Two active fractions around the 400 daltons following salt were eluted on Sephadex G-15 chromatography. The slower eluted factor competed kinetically with potassium ion, but the inhibitory activity was lost within two days during storage at 4 degrees C. The faster-eluted inhibitor lost its activity within a day. These results indicate that the unstable inhibiting factors of Na+, K+-ATPase exist in human urine and one of these factors inhibits ouabain sensitive Na+, K+-ATPase by binding to the potassium binding site (or very close to it), which exists at the outer surface of the cell membrane of this enzyme.