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Marinobufagenin (MBG) is an endogenous Na/K-ATPase inhibitor, a natriuretic and a vasoconstrictor. MBG is implicated in salt-sensitive hypertension, cardiac hypertrophy, and initiate the pro-fibrotic signaling. Previously it was demonstrated that immunoneutralization of an endogenous MBG by 3E9 anti-MBG-antibody (mAb) in vivo lowered blood pressure (BP) and reversed cardiac fibrosis in salt-sensitive, and in partially nephrectomized rats. In the present study, we investigated whether mAb alleviates vascular remodeling induced in normotensive rats on high salt intake.
Wistar rats (5 months old) received normal (CTRL; n = 8) or high salt intake (2% NaCl in drinking water) for 4 weeks ( n = 16). Rats from the group on a high salt intake were administered vehicle (SALT; n = 8) or mAb (50 µg/kg) (SALT-AB; n = 8) during the last week of high salt diet. BP, erythrocyte Na/K-ATPase activity, levels of MBG in plasma and 24-hour urine, and sensitivity of aortic explants to the vasorelaxant effect of sodium nitroprusside (SNP) were measured. Aortic collagen abundance was determined immunohistochemically.
In SALT vs. CTRL, heightened levels of MBG were associated with inhibition of erythrocyte Na/K-ATPase in the absence of BP changes. High salt intake was accompanied by a 2.5-fold increase in aortic collagen abundance and by a reduction of sensitivity of aortic explants to the vasorelaxant effect of SNP following endothelin-1-induced constriction. In the SALT-AB group, all NaCl-mediated effects were reversed by immunoneutralization of MBG.
High salt intake in young normotensive rats can induce vascular fibrosis via pressure-independent/MBG-dependent mechanisms, and this remodeling is reduced by immunoneutralization of MBG.
Marinobufagenin (MBG), an endogenous bufadienolide steroid, is a Na/K-ATPase inhibitor that participates in regulation of renal sodium transport and arterial blood pressure (BP).1–5 MBG promotes natriuresis via inhibition of sodium pump in the renal proximal tubules and vasoconstriction via inhibition of Na/K-ATPase in vascular smooth muscle cells.6–8 Endogenous Na/K-ATPase inhibitors, including MBG, become elevated in salt-sensitive hypertension,2–7 and it was demonstrated that angiotensin II stimulated production of MBG by adrenal cortex in salt-loaded rats.6 Inhibition of Na/K-ATPase in the smooth vasculature results in an increase of Ca2+ concentration through Na+/Ca2+ exchange,9 which further contributes to the MBG signaling.
It has been recently demonstrated that MBG initiates pro-fibrotic signaling via binding to Na/K-ATPase and activation of Src (sarcoma; proto-oncogene tyrosine-protein kinase) and EGFR (epidermal growth factor receptor) signaling resulting in degradation of Fli-1 (negative nuclear regulator of the procollagen-1 gene) in left ventricular myocardium, and induction of collagen-1 synthesis.10–12 Cardiac fibrosis was observed in rats administered MBG via minipumps, and in a rat model of uremic cardiomyopathy, in which endogenous MBG levels were concurrently elevated.13 Immunization of rats with 3E9 anti-MBG mAb reversed cardiac fibrosis in a model of renal failure,14 and reduced BP in models of preeclamsia and salt-sensitive hypertension.15,16 Increased plasma MBG levels in patients with preeclampsia17–19 are also accompanied by vascular fibrosis in umbilical preeclamptic arteries,19 and further, collagen-1 levels were increased in MBG-treated explants of umbilical arteries from women with uncomplicated pregnancies.19
In our previous study of a normal rat strain,20 high salt intake stimulated MBG production and tissue remodeling, in particular, in heart and kidney, and did not affect BP. We hypothesized that (i) a high salt intake in normotensive rats will stimulate the production of the endogenous Na/K-ATPase inhibitor MBG, resulting in induction of arterial fibrosis via a pressure-independent mechanism, and (ii) a monoclonal 3E9 anti-MBG antibody can alleviate this pro-fibrotic effect of MBG.
The protocol of the study was approved by the Animal Care and Use Committee of the National Institute on Aging. Prior to salt loading 5-month-old male Wistar rats were kept in a 26 °C environment with a 12:12 hour light–dark cycle on a normal NaCl diet (0.5%) and tap water ad libitum. Rats were divided into 3 groups: control group (CTRL; n = 8), NaCl intake (SALT; n = 8), NaCl intake plus administration of 3E9 anti-MBG-antibody (SALT-AB; n = 8). In SALT and SALT-AB groups tap water was substituted by a 2% NaCl solution for 4 weeks. Rats from the SALT-AB group were administered 3E9 anti-MBG antibody (mAb; total 50 μg/kg)16 intraperitoneally 3 times during the last week of salt loading. Body weight, 24-hour urine production, and BP were measured at baseline and at week 4 of the experiment. BP was recorded in the conscious state by tail-cuff plethysmography (IITC model 31; IITC Life Science, CA). Urine (24-hour) was collected in metabolic cages, and urine samples were kept frozen for subsequent measurement of Na+ and K+ and MBG (below). At the end of the experiment, rats were anesthetized by an intraperitoneal injection of 100mg/kg pentobarbital sodium and sacrificed by exsanguination from the abdominal aorta. Plasma samples were frozen for measurement of MBG (below). Na/K-ATPase activity was measured in erythrocytes (below). Thoracic aortae were removed immediately, the surrounding fat was removed, and the aortae were blotted and weighed. Wet aortic weight is presented as milligram per 100g of body weight. One part of each aorta was fixed in 4% formalin buffer solution for immunohistochemical analysis, and aortic rings were used to study the vasorelaxant effect of sodium nitroprusside (SNP).
Isolated rings of rat thoracic aortae 2.5–4.0mm in length were suspended at a resting tension of 1.5g in 15-ml organ bath (Ugo Basile, Varese, Italy) perfused by a solution containing (mmol/l): NaCl 130, KCl 4.0, CaCl2 1.8, MgCl2 1.0, NaH2PO4 0.4, NaHCO3 19, and glucose 5.4, and gassed with a mixture of 95% O2 and 5% CO2 (pH 7.45) as reported previously.19,21 Contraction force was recorded isometrically using force transducers based on KTD 2B tensoresistors. Aortic rings were constricted once with KCl (80 mmol/l), and after complete relaxation, the contractile response to the maximal effective concentration of endothelin-1 (100 nmol/l) was recorded, as in our previous study.19 Next, the ability of vessels to relax in response to incremental concentrations of SNP was assessed. The vasorelaxation response was expressed relative to plateau of contractile force that was achieved in response to endothelin-1. EC50 was calculated by nonlinear regression analysis of points producing 20% to 80% vasorelaxation and expressed in nmol/l.
Aliquots of the whole blood (0.5ml) were used to measure the Na/K-ATPase activity by the production of inorganic phosphate (Pi) in the presence and absence of 5 mmol/l ouabain, as described previously.16
Na+ and K+ concentrations in the plasma and urine were measured by 23Na NMR spectroscopy (Bruker Biospin, Billerica, MA) as reported previously,22 or by flame photometer technique (Bibby Scientific Limited, Staffordshire, UK). The total excretion of sodium and potassium ions is expressed as mmol/24h.
MBG was measured using fluoroimmunoassay (Dissociation-Enhanced FluoroImmunoAssay; DELFIA) based on a polyclonal rabbit anti-MBG-P antibody as reported previously.16 Renal MBG excretion is expressed as pmol/24h, plasma MBG concentration is given in nmol/l.
Aortic sections of 4–5mm in length were fixed in 4% formalin buffer solution for 12 hours, dehydrated and embedded in paraffin, then cut with a microtome. Thereafter, 5-µm-thick sections were stained with the collagen-specific dye Sirius Red/Fast Green Collagen Staining Kit (Chondrex, Redmond, WA). The collagen amount was estimated in the medial part of aorta by converting the pixels of each image with Metamorph Microscopy Automation and Image Analysis Software (Molecular Devices, LLC, CA) and expressed as percent of the total area.
Results are expressed as means ± SE and analyzed statistically using 1-way analysis of variance following Newman–Keuls test. P value less than 0.05 was considered statistically significant (Graph Pad Prism, Graph Pad Software, San Diego, CA).
BP did not differ between CTRL and SALT groups after 4 weeks of salt loading (Table 1 and Figure 1). Plasma levels and renal excretion of MBG in rats from SALT group were elevated compared to CTRL group (Figure 1A,,B).B). Compared to CTRL erythrocyte Na/K-ATPase activity in SALT group was decreased (Figure 1D), and sodium excretion in SALT group increased 15-fold (Table 1).
3E9 anti-MBG antibody (SALT-AB group) did not affect BP (Table 1 and Figure 1C), but reduced MBG in the urine (Figure 1B), and restored erythrocyte Na/K-ATPase activity vs. SALT group (Figure 1D). Interestingly, administration of 3E9 antibody resulted in additional increase in sodium excretion in SALT-AB group vs. SALT group (Table 1).
Data on the vasorelaxation of rat aortic rings, preconstricted with 100 nmol/l endothelin-1, are summarized in Figure 2 and Table 1. As shown in Figure 2A, aortic rings from CTRL, SALT, and SALT-AB groups exhibited similar sensitivity to maximal (100 nmol/l) concentration of endothelin-1. At the same time, the vasorelaxant effect of SNP was reduced in aortae from SALT group compared to CTRL (Figure 2B, Table 1). In SALT-AB group, sensitivity of aortic rings to vasorelaxant effect of SNP was restored to control levels (Figure 2B, Table 1). Aortic media thickness changed in neither SALT nor SALT-AB groups in comparison with CTRL group (Table 1). Aortic collagen deposition in SALT group was 2.6-fold higher than in CTRL (Figure 3A,,C)C) and was associated with increased wet aortic weight (Figure 3B, Table 1). Immunoneutralization of MBG with 3E9 anti-MBG antibody (SALT-AB group) reduced both the collagen abundance in the aortic wall (Figure 3A,,C)C) and aortic weight (Figure 3B) vs. SALT group.
The main novel finding of the present study is that in response to a high salt intake, an elevation of endogenous MBG level in young normotensive rats was accompanied by aortic remodeling in the absence of BP changes. Immunoneutralization of MBG by a monoclonal anti-MBG antibody during a high salt intake prevented aortic remodeling.
MBG is a natriuretic hormone which promotes natriuresis via inhibition of renal Na/K-ATPase.1–6 Elevated MBG levels are associated with an increased salt-sensitivity in animal models,2–7 and in offspring of rats NaCl-loaded throughout pregnancy.23 The present study demonstrates that elevated plasma levels and urinary MBG excretion in salt-loaded animals were accompanied by inhibition of erythrocyte Na/K-ATPase and increased sodium excretion. High salt intake did not affect BP in these normotensive rats, which is in accordance with our previous observations.15,20
Notably, the moderate elevation of MBG levels after 4 weeks of a high salt intake in the present study was accompanied by a substantial increase in collagen deposition in the aortic media. Previously we demonstrated that nanomolar MBG concentrations initiate myocardial pro-fibrotic signaling in a chronic renal failure model,13 and that in normotensive rats on a high NaCl intake,20 moderately increased levels of plasma and urinary MBG-induced left ventricular and renal remodeling. Moreover, incubation of rat aortic explants with nanomolar concentration of MBG ex vivo increases collagen abundance in the aortic media.21 This effect of MBG is based on its ability to inhibit Na/K-ATPase, which initiates the pro-fibrotic Fli-1-dependent signaling, resulting in the tissue remodeling.13 In rat aortic explants, preincubation with MBG was associated with an increased level of collagen in the aortic media and decreased vasorexation in response to SNP.21 In the present experiment, NaCl treatment for 4 weeks resulted in increased MBG level and collagen deposition in aortic media. An anti-MBG antibody blocked the circulating hormone MBG in vivo, which improved MBG-induced impairment of endothelium-independent vasorelaxation by SNP.
Our present findings are in accordance with our previous observation that heightened MBG levels after high salt intake are accompanied by pronounced fibrosis of the aortic wall. Here we demonstrated that immunoneutralization of elevated MBG in salt-loaded animals reduces aortic fibrosis, and therefore, that endogenous MBG can initiate arterial remodeling in normotensive rats. In addition, heightened MBG levels and increased aortic collagen abundance in rats after 4 weeks of high salt intake were associated with a marked reduction in the sensitivity of vascular rings to the vasorelaxant effect of SNP, in accordance with our previous observation on aortic rings that have been pre-incubated with MBG.21
The present study demonstrated that blunted vasorelaxation of aortic rings in response to a high salt intake was reduced by immunoneutralization of MBG. A similar observation was made previously in experiments in preeclamptic umbilical artery explants exposed in vitro to an anti-MBG monoclonal antibody, in which a preeclampsia-induced increase in vascular collagen abundance was reversed after MBG immunoneutralization.19 In the present study immunoneutralization of MBG in salt-loaded animals was associated with decreased renal MBG excretion and restoration of erythrocyte Na/K-ATPase, suggesting that in normotensive animals MBG initiates pro-fibrotic signaling via binding to Na/K-ATPase and that immunoneutralization of MBG reduces the arterial remodeling via preventing this binding. It might be interesting to study the pro-fibrotic effect of MBG in the small resistant arteries in the absence of hypertensive response in normotensive animals, which merits future investigation.
In our previous study we have demonstrated that high salt intake activated tissue renin-angiotensin system in Dahl salt-sensitive rats, and that heightened tissue angiotensin II stimulated adrenocortical MBG production in this model.6 Moreover, AT1 receptor blocker losartan prevented stimulation of MBG biosynthesis in vivo and in vitro.6 The recent publications also demonstrated a strong relationship between high salt intake and activation of renin-angiotensin system-dependent pro-fibrotic signaling, which cause the damage of cardiovascular and renal tissues in spontaneously hypertensive24 and normotensive rats.25,26 It is possible that in the present study in normotensive rats, renin-angiotensin system also may be activated by high dietary salt intake and thus contribute to the development of fibrosis. In the present study we have demonstrated that immunoneutralization of MBG was essential to prevent the fibrotic changes in aorta. Whether angiotensin II can also stimulate MBG production in these normotensive rats will be studied in the future. In the present study we have demonstrated that immunoneutralization of MBG was essential to prevent the fibrotic changes in aorta. Similar to our previous studies,16 immunoneutralization of MBG by 3E9 anti-MBG antibody in vivo was accompanied by increased sodium excretion (Table 1), the mechanism of which is unknown and merits further investigation. In the present experiment we did not study the collagen abundance, aortic weight, and aortic ability to relax in response to SNP at week 3 (before the antibody treatment), as well as the details of the pro-fibrotic signaling involved in vascular remodeling in normotensive animals, which will be investigated in our future studies.
In summary, we have demonstrated, for the first time that, in young normotensive rats, in response to a high salt intake, elevated MBG levels increased vascular fibrosis and induced impaired vasorelaxation in the absence of BP changes. Immunoneutralization of MBG reduced vascular fibrosis and improved vascular relaxation. Thus, high dietary NaCl intake can induce vascular fibrosis via pressure-independent/MBG-dependent mechanisms, and this remodeling reduced after immunoneutralization of MBG.
The authors declared no conflict of interest.
This work was supported by Intramural Research Program, National Institute on Aging, National Institutes of Health. We are grateful to Ruth Sadler for editorial assistance.