Sodium Pump Inhibitors In Blood Pressure Regulation

Aging

Investigators

Linked publications & trials

Abstract

Our previous studies demonstrated that two endogenous sodium pump ligands (SPL), endogenous ouabain (EO) and marinobufagenin (MBG), coexist in mammalian tissues. MBG acts as a selective inhibitor of ouabain resistant alpha-1 isoform of Na/K-ATPase (NKA), the main sodium pump isoform in the kidney, vascular smooth muscle and adult cardiomyocytes. In Dahl salt-sensitive rats (DS) on a high NaCl intake, brain EO triggers peripheral MBG, which raises the blood pressure. In a model of preeclampsia, pregnant Sprague-Dawley rats on a high NaCl intake raise their blood pressure in parallel with an increase in MBG excretion. In vivo, administration of polyclonal and monoclonal anti-MBG antibodies to hypertensive DS and pregnant rats with NaCl-induced hypertension, lowered the blood pressure. During the last year our research efforts concentrated on (i) the study of the relationship between central and peripheral SPLs in DS with chronic salt-sensitive hypertension, (ii) on the development of monoclonal anti-MBG antibodies as a potential treatment of preeclampsia, and (iii) on the studies of mechanisms of MBG-induced NKA-mediated cellular signaling underlying pathogenesis of NaCl-sensitive hypertension. (i) Pathogenesis of NaCl sensitive hypertension. Following acute NaCl loading of DS, an initial transient rise of EO in the hippocampus and amygdala, followed by an increase in EO in the supraoptic nucleus of the hypothalamus and pituitary, stimulates pituitary angiotensin II (ATII), and, via activation of sympathetic nervous system activates the renin-angiotensin system in the adrenal cortex. Adrenocortical ATII acting through AT1 receptors stimulates production of MBG. An increase in MBG production induces inhibition of the sodium pump in renal tubules and in cardiovascular tissues. During 2005 the above findings have been translated into the context of pathogenesis of chronic salt-sensitive hypertension in DS. In particular, administration of anti-ouabain antibody to DS prior to NaCl loading markedly reduces MBG response to salt loading and attenuates NaCl-induced hypertension. Moreover, administration of ouabain to DS with a normal NaCl intake produces a sustained increase in renal MBG excretion and causes hypertension. (ii) Monoclonal anti-MBG antibody as a therapy for preeclampsia. We developed three monoclonal anti-MBG antibodies (Mab) with high affinity to MBG and low cross reactivity to digoxin and ouabain. We tested the ability of Mab: (a) to lower blood pressure (BP) in vivo in hypertensive DS rats on a high NaCl intake, (b) to lower BP in rats with pregnancy-associated hypertension, and (c) to reverse the inhibition of NKA in erythrocytes from patients with preeclampsia. The effects of Mab were compared to that of DIGIBIND. In 12 DS following two weeks of 8% NaCl intake, in which renal MBG excretion increased 4-fold vs. baseline levels, a single intraperitoneal administration of Mab lowered BP by 40 mmHg and restored the Na/K-pump activity in thoracic aorta by 51%. Plasma MBG concentration in 10 patients with PE (systolic BP: 151?3 mm Hg vs. 99?3 mm Hg in normotensive control), measured by competitive immunoassay based on Mab, exhibited a ten-fold increase vs. that in 10 normotensive pregnant women. The activity of erythrocyte NKA in patients with PE was inhibited by 50% as compared to that in normotensive pregnant women. In vitro treatment of erythrocytes from PE patients with Mab restored the NKA activity (95% vs. control), while DIGIBIND was less effective (75% vs. control). NaCl supplementation of pregnant Sprague-Dawley rats was associated with a 37 mm Hg increase in systolic BP, a 3-fold rise in MBG excretion, and a 25% inhibition of the NKA in the thoracic aorta, as compared to pregnant rats on a normal NaCl intake. A single intraperitoneal administration of Mab to pregnant hypertensive rats reduced the BP (25 mm Hg) and restored the vascular Na/K-pump activity. (iii) MBG-induced cell signaling and mechanisms of its modulation. MBG is implicated in the pathogenesis of uremic cardiomyopathy (Priyadarshi, et al, KI 2003). Rats subjected to MBG infusion (10 ug/kg/day, SM) or partial nephrectomy (PNx) developed substantial cardiac fibrosis whereas PNx rats immunized against MBG (PNx-IM) were protected from this fibrosis. Western blot analyses of these tissues revealed 2-fold increases in fibronectin in SM and PNx without increases in TGFbeta, Smad 2/3, p-Smad 2/3 and Smad 4). The expression of Fli-1, a transcription factor, which inhibits collagen synthesis in dermal fibroblasts was decreased by 50% in both the SM and PNx hearts. In primary culture of rat fibroblasts grown to confluence, physiological concentrations of MBG added to the culture media increased the incorporation of proline into collagen, caused a 2-fold increase in ROS production as well as a 150% increase in procollagen expression over 24 hours. Fibroblast Fli-1 expression (Western blot) was decreased by 80% by 10 nM MBG. These data suggest that MBG produces cardiac fibrosis in a process involving increased ROS and decreased Fli-1 expression. In rat aorta and renal medulla we compared effects of ANP on NKA phosphorylation and on inhibition of NKA and sodium pump by MBG. MBG inhibited the NKA in renal and vascular membranes at the level of higher (nanomolar) and lower affinity (micromolar) binding sites. 1 nmol/L MBG inhibited the NKA in aortic sarcolemma (17%) and in renal medulla (20%). ANP potentiated MBG induced NKA inhibition in the kidney (49%), but reversed the effect of MBG in aorta. In a similar fashion, ANP modulated sodium pump inhibitory effects of MBG in rat aorta and in the proximal collecting tubules. In aorta, ANP dephosphorylated alpha-1 NKA, while in renal medulla ANP induced alpha-1 NKA phosphorylation. These effects of ANP on NKA phosphorylation and inhibition were mimicked by a protein kinase G (PKG) activator, 8-Br-PET-cGMP and prevented by a PKG inhibitor, KT5823. The fact, that in the kidney, inhibition of alpha-1 NKA by MBG is enhanced via NKA phosphorylation by ANP, while in the aorta ANP exerts an opposite effect, may be explained by expression of two different PKG isoforms in these tissues. The concurrent production of a vasorelaxant, ANP, and a vasoconstrictor, MBG, poteniate each other's natriuretic effects, but ANP peptides may offset the deleterious vasoconstrictor effect of MBG. Taken together, these findings demonstrate that MBG is an important factor in pathogenesis of hypertension, and open new pharmacological possibilities in the treatment of hypertension, including blockade of circulating MBG with a specific antibody and attenuation of NKA inhibitory effect of MBG on the NKA. In addition, MBG appears to contribute to cardiovascular remodeling via induction of cardiac fibrosis mediated through inhibition of Fli-1 expression.