Monoclonal Antibody to Marinobufagenin Downregulates TGFβ Profibrotic Signaling in Left Ventricle and Kidney and Reduces Tissue Remodeling in Salt-Sensitive Hypertension

Abstract

Background Elevated levels of an endogenous Na/K-ATPase inhibitor marinobufagenin accompany salt-sensitive hypertension and are implicated in cardiac fibrosis. Immunoneutralization of marinobufagenin reduces blood pressure in Dahl salt-sensitive (Dahl-S) rats. The effect of the anti-marinobufagenin monoclonal antibody on blood pressure, left ventricular (LV) and renal remodeling, and gene expression were investigated in hypertensive Dahl-S rats. Methods and Results Dahl-S rats were fed high NaCl (8%, HS; n=14) or low NaCl (0.1%, LS; n=14) diets for 8 weeks. Animals were administered control antibody (LS control antibody, LSC; HS control antibody, HSC; n=7 per group) or anti-marinobufagenin antibody once on week 7 of diet intervention (n=7 per group). Levels of marinobufagenin, LV, and kidney mRNAs and proteins implicated in profibrotic signaling were assessed. Systolic blood pressure was elevated (211±8 versus 133±3 mm Hg, P<0.01), marinobufagenin increased 2-fold in plasma (P<0.05) and 5-fold in urine (P<0.01), LV and kidney weights increased, and levels of LV collagen-1 rose 3.5-fold in HSC versus LSC. Anti-marinobufagenin antibody treatment decreased systolic blood pressure by 24 mm Hg (P<0.01) and reduced organ weights and level of LV collagen-1 (P<0.01) in hypertensive Dahl salt-sensitive rats with anti-marinobufagenin antibody versus HSC. The expression of genes related to transforming growth factor-β-dependent signaling was upregulated in the left ventricles and kidneys in HSC versus LSC groups and became downregulated following administration of anti-marinobufagenin antibody to hypertensive Dahl-S rats. Marinobufagenin also activated transforming growth factor-β signaling in cultured ventricular myocytes from Dahl-S rats. Conclusions Immunoneutralization of heightened marinobufagenin levels in hypertensive Dahl-S rats resulted in a downregulation of genes implicated in transforming growth factor-β pathway, which indicates that marinobufagenin is an activator of profibrotic transforming growth factor-β-dependent signaling in salt-sensitive hypertension.

Keywords: Dahl salt‐sensitive hypertension; cardiac hypertrophy; marinobufagenin; monoclonal antibody; transforming growth factor.

Figure 1

Changes in physiological and biochemical parameters in Dahl salt‐sensitive (Dahl‐S) rats on a high salt (HS) and low salt (LS) diet in the presence and absence of anti‐marinobufagenin antibody (mAb) treatment. A, Marinobufagenin in urine. B through D, Wet weight of left ventricles (LV), aortae, and kidneys correspondently, expressed per body weight (BW) at the end of week 8 of LS and HS diets in Dahl‐S rats treated with control antibody (LS control [LSC] and HS control [HSC]) and with anti‐marinobufagenin mAb (LSAB and HSAB, respectively), n=7 per group. Each bar represents the mean±standard error of the mean. By 1‐way ANOVA followed by Neuman–Keuls test: *P<0.05, **P<0.01, 1 of other groups vs LSC; ^†† P<0.01, HSAB vs HSC.

Figure 2

Left ventricle: Effect of a high salt (HS) diet and anti‐marinobufagenin antibody treatment on ERK1/2, transforming growth factor‐β1 (TGFβ‐1), Mothers Against DPP Homolog (SMAD) 4, mitogen‐activated protein kinase 3 (MAPK3), collagen‐1, and protein kinase C (PKC) δ protein abundance by Western blotting analysis in Dahl salt‐sensitive (Dahl‐S) rats. Top panels, Western blotting representative bands; bottom panels, statistical analysis of band density standardized for GAPDH for: (A) total ERK1/2; (B) TGFβ‐1 (total; mature form); (C) total SMAD4; (D) total MAPK3; (E) collagen‐1; (F) the ratio of phosphorylated PKCδ (p‐PKCδ) to total PKCδ protein; and (G) total Friend leukemia integration 1 transcription factor (Fli‐1) in the left ventricles of Dahl‐S rats. Each bar represents the mean±standard error of the mean of at least 3 measurements, n=7 per group. By 1‐way ANOVA followed by Neuman–Keuls post hoc test: *P<0.05, HS control [HSC] vs low salt control [LSC]; ^† P 0.05, high salt (HS) with anti‐marinobufagenin antibody (AB) (HSAB) vs HSC.

Figure 3

Left ventricle: effect of a high salt (HS) diet and anti‐marinobufagenin antibody treatment on mRNAs in Dahl salt‐sensitive (Dahl‐S) rats. A, Transforming growth factor (TGF) β‐1; (B) TGFβ‐2; (C) Mothers Against DPP Homolog (SMAD) 3; (D) SMAD4; (E) CTGF1; (F) COL1α2; (G) COL3α1; (H) COL4α1; and (I) COL5α1 mRNAs in the left ventricles of Dahl‐S by quantitative polymerase chain reaction analysis. Each bar represents the mean±standard error of the mean of 5 to 6 measurements, n=7 per group. By 1‐way ANOVA followed by Neuman–Keuls post hoc test: *P<0.05, **P<0.01, HS control [HSC] vs low salt control [LSC]; ^† P<0.05, ^†† P<0.01, high salt (HS) with anti‐marinobufagenin antibody (AB) (HSAB) vs HSC.

Figure 4

Renal medulla: Effect of a high salt (HS) diet and anti‐marinobufagenin antibody treatment on renal transforming growth factor (TGF) β‐1 (total; mature form) and collagen‐1 protein abundance by Western blotting analysis in Dahl salt‐sensitive (Dahl‐S) rats. Top: Western blotting representative bands. Bottom: Statistical analysis of band density standardized for GAPDH. A, TGFβ‐1; and (B) collagen‐1 in the renal medulla of Dahl‐S rats. Each bar represents the mean±standard error of the mean of at least 3 measurements, n=7 per group. By 1‐way ANOVA followed by Neuman–Keuls post hoc test: *P<0.05, HS control [HSC] vs low salt control [LSC]; ^† P<0.05, high salt (HS) with anti‐marinobufagenin antibody (AB) (HSAB) vs HSC.

Figure 5

Renal medulla: Effect of a high salt (HS) diet and anti‐marinobufagenin antibody treatment on renal mRNAs. A, Transforming growth factor (TGF) β‐1; (B) fibronectin 1 (FN1); (C) mitogen‐activated protein kinase 1 (MAPK1); (D) COL1α2; (E) COL3α1; and (F) COL4α1 in Dahl salt‐sensitive rats by quantitative polymerase chain reaction analysis. Each bar represents the mean±standard error of the mean of at least 5 measurements, n=7 per group. By 1‐way ANOVA followed by Neuman–Keuls post hoc test: *P<0.05, **P<0.01 one of the other groups vs low salt control [LSC]; ^† P<0.05, ^†† P<0.01, high salt (HS) with anti‐marinobufagenin antibody (AB) (HSAB) vs HS control (HSC).

Figure 6

Effect of a high salt (HS) diet and anti‐marinobufagenin antibody treatment on the Heart Failure Network in the left ventricles of Dahl salt‐sensitive rats. Gene expression network profile shows genes in the Heart Failure Network that were upregulated (red) or downregulated (green) in left ventricular (LV) tissue HS control [HSC] vs low salt control [LSC] groups (A), and high salt (HS) with anti‐marinobufagenin antibody (AB) (HSAB) vs HSC groups (B). Green and red indicate significant (P<0.05) changes in gene expression between indicated group pairs; intensity of color indicates the magnitude of Z‐ratio between groups (the darkest color is for the highest Z‐ratio).

Figure 7

Profibrotic effect of marinobufagenin on cultured left ventricular myocytes of Dahl salt‐sensitive rats kept on a low salt (LS) diet (n=6). Addition of marinobufagenin (1 and 10 nmol/L) for 24 hours to the culture media–activated production of: (A) transforming growth factor (TGF) β‐1 (mature form); (B) Mothers Against DPP Homolog (SMAD) 2; (C) mitogen‐activated protein kinase (MAPK) 42/44; (D) collagen‐1; (E) increased amount of phosphorylated protein kinase C (PKC) δ, and (F) decreased level of Friend leukemia integration 1 transcription factor (Fli‐1). Top panels: representative Western blotting images; bottom panels: statistical analysis of band density for each protein standardized for GAPDH. Each bar represents the mean±standard error of the mean; each protein was tested at least 5 times for each condition. By 1‐way ANOVA followed by Neuman–Keuls post hoc test: *P<0.05, **P<0.01 one of the other groups vs baseline (0 nmol/L marinobufagenin); ^† P<0.05, 10 nmol/L vs 1 nmol/L marinobufagenin.

Figure 8

Schematic presentation of signaling (non‐Mothers Against DPP Homolog [SMAD] and SMAD‐dependent) pathways initiated by marinobufagenin via interaction with Na/K‐ATPase. ANGII indicates angiotensin II; c‐Src, proto‐oncogene tyrosine‐protein kinase; EGFR, epidermal growth factor receptor; ERK1/2, mitogen‐activated protein kinase (MAPK); Fli‐1, Friend leukemia integration 1 transcription factor, a negative regulator of collagen‐1 production; NKA, Na/K‐ATPase; PLCγ, phospholipase C gamma; PKCδ, protein kinase C delta; TGFβ, transforming growth factor‐β; TGFβR, TGFβ receptor. The dotted line indicates the release of the procollagen DNA promoter by phosphorylated Fli1; “P” in yellow circle, phosphorylated form of the protein. The chemical structure of marinobufagenin (MBG) is given in the insertion on the left.