Preeclampsia (PE) is a serious pregnancy complication which is characterized by severe hypertension. MBG levels increase in plasma and placentae of PE patients in the presence of inhibited Na/K-ATPase in erythrocytes. We have demonstrated that MBG initiates a pro-fibrotic Fli-1 -dependent signaling via binding to Na/K-ATPase and activation of EGFR, which initiates increase in collagen-I production PE (Nikitina et al. J Hypertens. 2011). Recently we have found in addition to Fli-1-dependent pathway, heightened MBG levels in PE may be responsible for phosphorylation and activation of the key members of another pro-fibrotic pathways, ERK1/2 and PKC-delta, downstream of EGFR-dependent signaling. We also demonstrated that in addition to collagen-I, levels of collagen-IV increased in placentae from PE patients vs. tissues from control subjects with normal pregnancies, which may be due to activation of this pro-fibrotic signaling. This finding was made after the incubation of placental explants from PE patients with monoclonal anti-MBG antibody 3E9 (mAb). Western blotting analysis demonstrated that ratio of phosphorylated forms of ERK1/2 (pERK1/2) and PKC-delta (pPKC-delta) to the total protein amount was increased in PE placental explants vs. that in the placental explants from the non-complicated pregnancy. Incubation of the placental explants with anti-MBG 3E9 mAb for 24 hours in the culture media was associated with a decrease in the levels of both pERK1/2 and pPKC-delta in PE placenta sample vs. vehicle-treated PE placental explants. Thus, ex vivo immunoneutralization of MBG was associated with down-regulation of two key players in the pro-fibrotic signaling. MBG induces fibrosis via inhibition of Fli1, a nuclear transcription factor and a negative regulator of collagen synthesis. Because in uremic rats immunization against MBG reduced cardiac fibrosis but minimally affected BP, we hypothesized that MBG induces cardiovascular fibrosis via BP-independent mechanism. We determined BP, plasma and urinary MBG, aortic collagen-1, and vascular function in NaCl-loaded male Wistar rats with type 2 diabetes mellitus (DM-NaCl) and in control (CTRL) rats. DM was induced by a single administration of 65 mg/kg streptozotocin to neonatal animals. At 3 months of age, 24 rats were NaCl supplemented and drank 1.8% NaCl for four weeks and were twice administered i/p vehicle (n=12) or 3E9 mAb (n=12) during last week of NaCl loading. Isolated rings of thoracic aortae were tested for their responsiveness to endothelin-1 and to sodium nitroprusside (SNP) following endothelin-1-induced constriction. DM-NaCl rats exhibited a 3.5-fold increase in MBG excretion (7.7 +/- 1.5 nmoles vs. 25.4 +/- 4.5 nmoles in CTRL; P<0.001) and 2.5-fold increase in levels of collagen-1 in thoracic aortae vs. CTRL without changes of BP and renal function. As compared to CTRL, aortic rings from DM-NaCl rats exhibited unaltered responsiveness to endothelin-1 (EC50 = 2.2 and 3.2 nmol/L, respectively), but impaired response to the relaxant effect of SNP (EC50= 29 nmol/L vs. 7 nmol/L P<.001). In vivo administration of 3E9 mAb to DM-NaCl rats did not affect BP, but reduced aortic levels of collagen-1, and restored sensitivity of aortic rings to SNP (EC50 = 9 nmol/L). Thus, MBG induces vascular fibrosis and increases vascular stiffness without affecting BP in rat model of DM. Next we studied the interaction of mineralocorticoid receptor (MR) antagonists and MBG on Na/K-ATPase. Previously we have demonstrated that rat model of chronic renal failure (partial nephrectomy (PNx) model) is characterized by elevated MBG levels and renal and cardiovascular tissue fibrosis. Administration of MR antagonist spironolactone to these rats reversed tissue fibrosis and reduced BP. We have estimated effect of MBG on Na/K-ATPase from rat kidney in the absence and presence of canrenone, an active metabolite of spironolactone. MBG inhibited Na/K-ATPase from rat renal medulla in a concentration-dependent fashion, and addition of 10 mol/L canrenone to the incubation medium markedly reduced sensitivity of the Na/K-ATPase to MBG (IC50 = 1.9 +/- 0.5 mol/L and 113+/- 11 mol/L). Because activity of erythrocyte Na/K-ATPase could be a marker for circulating MBG activity, we next tested Na/K-ATPase inhibitory effect of MBG in the absence and in the presence of canrenone in rat erythrocytes in vitro. Canrenone alone did not alter erythrocyte Na/K-ATPase activity, but reversed the inhibitory effects of 100 nmol/L MBG. Thus, MR antagonists are effective modulators of inhibitory activity of MBG in vitro and in vivo. Experimental uremic cardiomyopathy causes cardiac fibrosis, and is causally related to the increased circulating levels of the MBG. The mammalian target of rapamycin is an mTOR, a serine/threonine kinase implicated in the progression of many different forms of renal disease. Rapamycin also inhibits activity of cytochrome P450 enzyme CYP27A1, which initiated biosynthesis of MBG. Treatment with rapamycin has been shown to attenuate fibrosis in experimental models of renal disease. As Na/K-ATPase signaling is known to stimulate the mTOR system, we speculated that the ameliorative effects of rapamycin might influence this pathway. Male Sprague Dawley rats were subjected to either PNx, infusion of MBG, and/or infusion of rapamycin through osmotic minipumps. The PNx animals showed marked increase in plasma MBG levels (1026 +/- 20 vs. 378 +/- 18 pmol/L, P<0.01), systolic BP (169 +/- 1 vs. 111 +/- 1 mmHg, p<0.01), and cardiac fibrosis compared to controls. Plasma MBG levels were significantly decreased in the PNx-rapamycin animals compared to PNx (372 +/- 15 vs 1026 +/- 20 pmol/L, P<0.01), and cardiac fibrosis was substantially attenuated by rapamycin treatment. Conclusion: In agreement with our previous findings, these observations further indicate causative link between elevated levels of an endogenous MBG and vascular fibrosis in animal models of chronic renal failure and hypertension. Anti-MBG monoclonal antibody, rapamycin, which blocks MBG biosynthesis, and canrenone, which disrupts interaction between MBG and Na/K-ATPase, exhibit beneficial effects in experimental hypertension and renal failure.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAAG000869-09
Application #
9351976
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Aging
Department
Type
DUNS #
City
State
Country
Zip Code
Fedorova, Olga V; Ishkaraeva, Valentina V; Grigorova, Yulia N et al. (2018) Antibody to Marinobufagenin Reverses Placenta-Induced Fibrosis of Umbilical Arteries in Preeclampsia. Int J Mol Sci 19:
Strauss, Michél; Smith, Wayne; Wei, Wen et al. (2018) Marinobufagenin is related to elevated central and 24-h systolic blood pressures in young black women: the African-PREDICT Study. Hypertens Res 41:183-192
AlGhatrif, Majd; Wang, Mingyi; Fedorova, Olga V et al. (2017) The Pressure of Aging. Med Clin North Am 101:81-101
Gable, Marjorie E; Ellis, Linda; Fedorova, Olga V et al. (2017) Comparison of Digitalis Sensitivities of Na(+)/K(+)-ATPases from Human and Pig Kidneys. ACS Omega 2:3610-3615
Haller, Steven T; Yan, Yanling; Drummond, Christopher A et al. (2016) Rapamycin Attenuates Cardiac Fibrosis in Experimental Uremic Cardiomyopathy by Reducing Marinobufagenin Levels and Inhibiting Downstream Pro-Fibrotic Signaling. J Am Heart Assoc 5:
Fedorova, Olga V; Emelianov, Igor V; Bagrov, Konstantin A et al. (2015) Marinobufagenin-induced vascular fibrosis is a likely target for mineralocorticoid antagonists. J Hypertens 33:1602-10
Grigorova, Yulia N; Juhasz, Ondrej; Zernetkina, Valentina et al. (2015) Aortic Fibrosis, Induced by High Salt Intake in the Absence of Hypertensive Response, Is Reduced by a Monoclonal Antibody to Marinobufagenin. Am J Hypertens :
Fedorova, Olga V; Zernetkina, Valentina I; Shilova, Victoria Y et al. (2015) Synthesis of an Endogenous Steroidal Na Pump Inhibitor Marinobufagenin, Implicated in Human Cardiovascular Diseases, Is Initiated by CYP27A1 via Bile Acid Pathway. Circ Cardiovasc Genet 8:736-45
Fedorova, Olga V; Lakatta, Edward G; Bagrov, Alexei Y et al. (2015) Plasma level of the endogenous sodium pump ligand marinobufagenin is related to the salt-sensitivity in men. J Hypertens 33:534-41; discussion 541
Kennedy, David J; Shrestha, Kevin; Sheehey, Brendan et al. (2015) Elevated Plasma Marinobufagenin, An Endogenous Cardiotonic Steroid, Is Associated With Right Ventricular Dysfunction and Nitrative Stress in Heart Failure. Circ Heart Fail 8:1068-76

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