Background — Digitalis-like sodium pump ligands (SPLs) effect natriuresis via inhibition of renal tubular Na ⁺ ,K ⁺ -ATPase but may induce vasoconstriction. The present study investigated the potential roles of 2 putative endogenous SPLs, an ouabain-like compound (OLC) and an α ₁ Na ⁺ ,K ⁺ -ATPase inhibitor, marinobufagenin (MBG), in regulating natriuresis and blood pressure (BP) responses to sustained and acute NaCl loading in Dahl salt-sensitive rats (DS). Methods and Results — During 4 weeks of an 8% NaCl diet, DS exhibited a progressive increase in MBG renal excretion (66±13 pmol/24 hours at week 4 versus 11±1 pmol/24 hours at baseline, n=48), which paralleled an increase in systolic BP (174±10 mm Hg at week 4 versus 110±2 mm Hg at baseline). By contrast, OLC excretion peaked at week 1 and returned to baseline levels. Administration of an anti-MBG, but not anti-ouabain antibody, to DS after 3 weeks of a high NaCl diet lowered BP (139±7 versus 175±5 mm Hg, P <0.001, n=5). Acute NaCl loading (2 hours) of DS (n=5) increased MBG and OLC excretion and natriuresis. Pretreatment of acutely NaCl-loaded DS with an anti-MBG antibody (n=5) reduced the excretion of sodium and MBG but not that of OLC. An anti-ouabain antibody (n=5) reduced sodium excretion and both OLC and MBG. Conclusions — An initial transient stimulation of OLC induced by NaCl loading of DS precedes an MBG response. A sustained increase in MBG production in DS contributes to the chronic BP elevation induced by a sustained high NaCl intake.

Fluoride (F) is ubiquitous natural substance and widespread industrial pollutant. Although low fluoride concentrations are beneficial for normal tooth and bone development, acute or chronic exposure to high fluoride doses results in adverse health effects. The molecular mechanisms underlying fluoride toxicity are different by nature. Fluoride is able to stimulate G-proteins with subsequent activation of downstream signal transduction pathways such as PKA-, PKC-, PI3-kinase-, Ca²⁺-, and MAPK-dependent systems. G-protein-independent routes include tyrosine phosphorylation and protein phosphatase inhibition. Along with other toxic effects, fluoride was shown to induce oxidative stress leading to excessive generation of ROS, lipid peroxidation, decrease in the GSH/GSSH ratio, and alterations in activities of antioxidant enzymes, as well as to inhibit glycolysis thus causing the depletion of cellular ATP and disturbances in cellular metabolism. Fluoride triggers the disruption of mitochondria outer membrane and release of cytochrome c into cytosol, what activates caspases-9 and -3 (intrinsic) apoptotic pathway. Extrinsic (death receptor) Fas/FasL-caspase-8 and -3 pathway was also described to be implicated in fluoride-induced apoptosis. Fluoride decreases the ratio of antiapoptotic/proapoptotic Bcl-2 family proteins and upregulates the expression of p53 protein. Finally, fluoride changes the expression profile of apoptosis-related genes and causes endoplasmic reticulum stress leading to inhibition of protein synthesis.

Dahl salt-sensitive rats (DS), which have a mutation in the α-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 α-1 than α-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 α-1 Na ⁺ /K ⁺ -ATPase isoform). Unlike ouabain (IC ₅₀ =248 μmol/L), serially diluted, HPLC-purified MBG immunoreactivity from DS and authentic MBG potently inhibited rat kidney Na ⁺ /K ⁺ -ATPase (IC ₅₀ =70 and 78 nmol/L, respectively). Our results suggest that an α-1 Na ⁺ /K ⁺ -ATPase ligand, MBG, is elaborated to promote natriuresis in hypertensive DS. MBG acts as a selective inhibitor of the ouabain-resistant α-1 Na ⁺ /K ⁺ -ATPase subunit, ie, the major sodium pump isoform of the kidneys, as would be expected of a putative natriuretic hormone.

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 μg/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 μmol 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.

The pathogenesis of pre-eclampsia (PE), a major cause of maternal and fetal mortality, is not fully understood. Digitalis-like sodium pump ligands (SPLs) are believed to be implicated in PE, as illustrated by clinical observations that DIGIBIND, a digoxin antibody that binds SPLs, lowers blood pressure (BP) in PE. We recently reported that plasma levels of marinobufagenin (MBG), a vasoconstrictor SPL, are increased four-fold in patients with PE. In the present study, we tested whether a polyclonal antibody to MBG can lower BP in rats with pregnancy-associated hypertension. Systolic BP (SBP), 24-h renal excretion of MBG and endogenous ouabain (EO), and sodium pump activity in the thoracic aortae were measured in virgin and pregnant Sprague–Dawley rats without and with NaCl supplementation (drinking 1.8% NaCl solution). NaCl supplementation of virgin rats stimulated renal excretion of MBG by 60%, but not that of EO, and did not change the BP. Compared with virgin rats, the last week of pregnancy in non-NaCl-loaded rats was associated with a decrease in SBP (106 ± 2 versus 117 ± 2 mmHg); a moderate increase in renal excretion of MBG (97.6 ± 4.9 versus 57.4 ± 7.0 pmoles/24 h) and EO (36.2 ± 4.3 versus 24.1 ± 3.2 pmoles/24 h). NaCl-loaded pregnant rats exhibited elevation in SBP (139 ± 3 mmHg; P < 0.01 versus non-NaCl-loaded pregnant rats), in renal excretion of MBG (160.0 ± 17.5 pmoles/24 h; P < 0.01 versus non-NaCl-loaded pregnant rats), but not in EO, and showed fetal growth retardation. Administration of the anti-MBG antibody to NaCl-loaded pregnant rats lowered SBP (111 ± 2 mmHg; P < 0.01) and increased aortic sodium pump activity (144 ± 3 versus 113 ± 5 nmol 86Rb/g per min; P < 0.01 versus non-NaCl-loaded pregnant rats). These observations provide evidence that MBG contributes to BP elevation in pregnant rats rendered hypertensive by NaCl supplementation.

In NaCl-loaded Dahl salt-sensitive (DS) rats the transient stimulation of brain endogenous ouabain (EO) precedes the increase in renal excretion of marinobufagenin (MBG), a vasoconstrictor and natriuretic. In hypertensive DS rats, EO raises blood pressure (BP) via an ATII-sensitive pathway. We hypothesized that an NaCl-induced increase in MBG is linked to the EO-stimulated ATII pathway. We studied the effects of 3 h of NaCl loading (17 mmol/kg, intraperitoneally) in male DS rats treated with antibodies to MBG or ouabain, or with losartan (25 mg/kg). NaCl loading alone induced a transient stimulation of pituitary EO (22.4 ± 1.8 versus 12.2 ± 1.3 pmol/g) and ATII (39.4 ± 2.8 versus 18.4 ± 3.2 ng/g), a sustained increase in MBG excretion (5.2 ± 0.6 versus 1.1 ± 0.2 pmol/h), a 40% inhibition of the renal sodium pump, a natriuretic response, a 35 mmHg increase in systolic BP, and an increase in adrenocortical ATII and MBG levels and in plasma norepinephrine. The anti-MBG antibody reduced the natriuresis (36%) and BP (40 mmHg), and restored renal sodium pump activity. The anti-ouabain antibody prevented the increase in pituitary ATII, reduced MBG excretion, natriuresis and BP, increased sodium pump activity, and prevented increases in plasma norepinephrine, pituitary and adrenocortical ATII, and adrenocortical MBG. Losartan mimicked the effects of the anti-ouabain antibody, but did not affect the excretion of EO. In adrenocortical cells of DS rats, ATII stimulated MBG secretion, and losartan blocked this effect. In response to NaCl loading, brain EO, via an AT1 receptor pathway and probably via sympathetic activation, stimulates adrenocortical MBG, which inhibits the renal sodium pump and elevates BP.