Learn more: PMC Disclaimer | PMC Copyright Notice
Monoclonal antibody against marinobufagenin reverses cardiac fibrosis in rats with chronic renal failure
Abstract
Background
Cardiotonic steroids (CTS) are implicated in pathophysiology of uremic cardiomyopathy. In the present study, we tested whether a monoclonal antibody (mAb) against the bufadienolide CTS, marinobufagenin (MBG), alleviates cardiac hypertrophy and fibrosis in partially nephrectomized (PNx) rats.
Methods
In PNx rats, we compared the effects of 3E9 anti-MBG mAb and of Digibind, an affinity-purified digoxin antibody, on blood pressure and cardiac hypertrophy and fibrosis following 4 weeks after the surgery.
Results
In PNx rats, a four-fold elevation in plasma MBG levels was associated with hypertension, increased cardiac levels of carbonylated protein, cardiac hypertrophy, a reduction in cardiac expression of a nuclear transcription factor which is a negative regulator of collagen synthesis, Fli-1, and an increase in the levels of collagen-1. A single intraperitoneal administration of 3E9 mAb to PNx rats reduced blood pressure by 59 mmHg for 7 days and produced a significant reduction in cardiac weight and cardiac levels of oxidative stress, an increase in the expression of Fli-1, and a reduction in cardiac fibrosis. The effects of Digibind were similar to those of 3E9 mAb, but were less pronounced.
Conclusions
In experimental chronic renal failure, elevated levels of MBG contribute to hypertension and induce cardiac fibrosis via suppression of Fli-1, representing a potential target for therapy.
Introduction
Uremic cardiomyopathy is a major cause of morbidity and mortality in patients with chronic kidney disease.1 Despite considerable recent progress in the understanding of the pathogenesis of uremic cardiomyopathy, there is clearly a niche for novel approaches to its treatment.1,2 An increasing body of evidence indicates that one of the factors implicated in pathogenesis of uremic cardiomyopathy is the group of hormones known as endogenous cardiotonic steroids (CTS).3 CTS regulate sodium pump activity at a cellular level and are implicated in the regulation of natriuresis and vascular tone.3 Many of the effects of these hormones appear to derive from a signaling function of the Na/KATPase; in particular, this signaling stimulated by CTS leads to cardiac hypertrophy and fibrosis.4,5 Previously we demonstrated that circulating concentrations of marinobufagenin (MBG) (14,15β-Epoxy-3β,5-dihydroxy-5β-bufa-20,22-dienolide), an endogenous bufadienolide CTS, are elevated in patients with renal failure and in partially nephrectomized rats (PNx).5,6 In PNx rats, also we observed increased cardiac and plasma levels of carbonylated proteins as well as other evidence for signaling through the Na/K-ATPase such as activation of Src and MAPK.5,6 In these studies, active immunization of PNx rats against MBG dramatically reduced cardiac hypertrophy and fibrosis and systemic oxidant stress, as well as evidence of Na/K-ATPase signaling. Conversely, chronic administration of MBG to normotensive rats to achieve similar plasma concentrations of MBG as seen with PNx produced a very similar cardiac phenotype similar to PNx.5,6
The transcription factor, Friend leukemia integration-1 (Fli-1), a member of the ETS family, is a negative regulator of collagen synthesis,7 and reduced levels of Fli-1 were documented in skin fibroblasts of patients with scleroderma.8,9 Recent evidence indicates that suppression of Fli-1 is also implicated in profibrotic signaling by CTS. In vitro, we have demonstrated that nanomolar concentrations of MBG stimulate collagen production by dermal, cardiac, and renal fibroblasts by a mechanism involving PKC-δ-dependent phosphorylation and depletion of Fli-1.7 Interestingly, when we stably transfected renal fibroblasts with a Fli-1 expression vector which dramatically increased Fli-1 expression, the basal expression of procollagen was decreased and MBG treatment did not increase procollagen expression or appreciably reduce Fli-1 expression.7
Recently, we developed two anti-MBG monoclonal antibodies (mAb), 3E9 and 4G4.10 In our previous experiments 3E9 mAb exceeded 4G4 with respect to reversal of MBG-induced Na/K-ATPase inhibition, and potently reduced blood pressure and restored vascular sodium pump activity in hypertensive Dahl-S rats and in pregnant Sprague-Dawley rats rendered hypertensive by NaCl supplementation. Because of these properties, in the present experiment we used 3E9 mAb for in vivo MBG immunoneutralization, while 4G4 mAb which exhibits high affinity to MBG in competitive immunoassays was chosen for MBG measurement.10 In the present experiment, in PNx rats, we studied effects of 3E9 anti-MBG mAb on arterial pressure, cardiac fibrosis and oxidative stress, and cardiac expression of Fli-1. We also compared effects of 3E9 mAb to those of Digibind (the Fab fragments of ovine digoxin antibody) which has been demonstrated to both bind endogenous CTS11, as well as lower blood pressure in patients with preeclampsia,12,13 a clinical syndrome known to have elevated CTS levels.10,14
Materials and methods
Animal studies
All animal experimentation described in this article was conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals under protocols approved by the University of Toledo Institutional Animal Care and Use Committee. Male Sprague Dawley rats (250–300 grams) were used for these studies. Eight sham-nephrectomized rats comprised the control group. In 18 rats, PNx (5/6 nephrectomy) was produced by surgical removal of the right kidney and ligation of the two-thirds of the arterial supply to the left kidney as reported previously in detail.15 In brief, rats were anesthesized with a mixture of 100% oxygen and 5% isoflurane, an incision was made in the left flank, through which the left kidney was pulled out, and arteries supplying to upper and lower poles were ligated. After a week, the right kidney was decapsulated to avoid removal of adrenal gland, artery, vein and ureter were ligated, and the kidney was removed. This maneuver produces sustained hypertension within 2 weeks.5–6 At four weeks following PNx, these rats intraperitoneally administered vehicle (n=6), Digibind (n=6) or 3E9 anti-MBG mAb (n=6). The dose of Digibind (10 µg/kg) was similar to that previously administered to patients with preeclampsia,12,13 and the dose of 3E9 Mab (50 µg/kg) was the same as that previously reported to reverse the EC75 to the inhibition of the Na/K-ATPase by MBG in rat renal outer medulla in vitro, and to reduce blood pressure in hypertensive Dahl-S rats in vivo.10 Blood pressure was determined using the tail cuff method by IITC, Inc. (Amplifier model 229, Monitor model 31, Test chamber Model 306; IITC Life Science, Woodland Hills, CA) at baseline, 3, 24, 48 hrs and at 1 week following antibody treatment (5 weeks post PNx). Then rats were sacrificed and the heart weight and cardiac histology were determined. Plasma samples were stored at −80°C for determination of CTS.
Oxidative stress markers
Levels of oxidative stress were assessed by measurement of protein carbonyl levels and determination of intracellular production of reactive oxygen species using the fluorescent probe dye dihydroethidium (DHE). Total protein carbonyl concentration of the plasma and left ventricular homogenate was determined by ELISA using the BIOCELL PC Test kit (Northwes Life Science Specialties). Production of reactive oxygen species was detected by DHE (Invitrogen Molecular Probes, Eugene, OR, USA) as described previously.16,17 Briefly, left ventricle tissue was frozen in OCT compound, and transverse sections (10μm) were generated with a cryostat and placed on glass slides. Tissue sections were incubated with 5uM DHE at 37°C for 20 min according to the manufacturer’s instructions. Red fluorescence was assessed by using an Olympus FSX100 box type fluorescence imaging device (Olympus America Inc., Center Valley, PA., USA). The excitation wavelength was 488 nm with emission at 585 nm. Fluorescence intensity was analyzed by the use of Image J (version 1.32j) software (National Institutes of Health, USA http://rsb.info.nih.gov/ij/).
Creatinine and creatinine clearance
At the conclusion of the study, 24hr urine samples were collected. At the end of urine collection, animals were sacrificed and blood samples were obtained from abdominal aorta. Plasma creatinine was measured with a colorimetric method using a commercial kit from Teco Diagnostics (Anaheim, CA, Cat# C515-480). Creatinine standards or plasma samples were mixed with the picric acid reagent and creatinine buffer reagent provided with the kit. The OD value at 510 nm was measured immediately after and at 15 min. The differences between the two time points were used to calculate the creatinine concentrations. Creatinine clearance was calculated using the following formula: [urine Cr×urine Vol (ml) / plasma Cr × 24hrs × 60 min].
Western blot analyses of Fli-1 and collagen-1
Western Blot analysis was performed on proteins from tissue homogenates as previously reported.7 The left ventricles from the heart were homogenized in ice-cold RIPA lysis buffer (pH 7.0) Santa Cruz (sc-24948). The homogenate was centrifugated at 1400 G for 30 sec at 4°C. The supernatant was discarded and the pellet fraction was re-suspended in 5% SDS and 50 mM Tris-HCl (pH 7.4). The protein was quantified in the resuspended pellet fraction and was solubilized in sample buffer (2% SDS, 5% β-mercaptoethanol, 20% glycerol, 0.005% bromophenol blue and 50mM Tris-HCl pH 7.0). The proteins, obtained from tissue homogenates, were resolved on an SDS-PAGE using Precast Ready Gels 4–15 % Tris-HCl, purchased from Bio-Rad (Hercules,CA). Ten micrograms of protein per sample were loaded into each well. The proteins from the gel were electrotransferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in 20 mM Tris-HCl (pH 7.5, 150 mM NaCl, and 0.1% Tween 20). Goat anti-type1collagen antibody (Southern Biotech, Birmingham, AL) was used to probe for collagen-1, and secondary anti-goat antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). To probe for Fli-1, we used rabbit polyclonal anti-Fli1(C19) antibody (Santa-Cruz Biotechnology; 1:500) and peroxidase-conjugated antirabbit antiserum (Amersham Corp., 1:1000). For detection, we used ECL and ECL plus purchased from Amersham Biosciences (Piscataway, NJ). Loading conditions were controlled for using actin (mouse monoclonal antibody, Santa Cruz).
Histology
Trichrome staining was performed on left ventricular tissues and tissue fibrosis was quantified as previously reported.6,7,15 Left ventricle sections were immediately fixed in 4% formalin buffer solution (pH 7.2) for 18 h, dehydrated in 70% ethanol, and then embedded in paraffin and cut with a microtome. Trichrome staining was then performed and fibrosis was quantified using ImageJ software. For quantitative morphometric analysis, five random sections of trichrome slides were electronically scanned into an RGB image which was subsequently analyzed using Image J (version 1.32j) software (National Institutes of Health, USA http://rsb.info.nih.gov/ij/). The amount of fibrosis was then estimated from the RGB images with a macro written by the authors (JIS) by converting pixels of the image with substantially greater (> 120%) blue than red intensity to have the new, grey scale amplitude = 1, leaving other pixels as with amplitude = 0
MBG immunoassay
For measurement of MBG, plasma samples were extracted using C18 SepPak cartridges (Waters Inc, Cambridge, USA). Cartridges were activated with 10 ml acetonitrile and washed with 10 ml water. Then 0.5 ml plasma samples were applied to the cartridges and consecutively eluted in the same vial with 7 ml 20% acetonitrile followed by 7 ml 80% acetonitrile and vacuum dried. Before immunoassays, samples were reconstituted in the initial volume of assay buffer. MBG was measured using a fluoroimmunoassay based on a murine anti-MBG 4G4 mAb recently described in detail.10 This assay is based on competition between immobilized antigen (MBG-glycoside-thyroglobulin) and MBG or other cross-reactants, within the sample for a limited number of binding sites on 4G4 anti-MBG mAbs. Secondary (goat anti-mouse) antibody labeled with nonradioactive europium was obtained from Perkin-Elmer (Waltham, Massachusetts, USA). Data on cross-reactivity of the 4G4 mAb used for determination of MBG levels and of 3E9 mAb used for in vivo administration were reported previously in detail.10 MBG (>98% HPLC pure) was purified from secretions from parotid glands of Bufo marinus toads as reported previously.10
Statistical analyses
The results are presented as means ± SEM. Data were analyzed using one-way ANOVA followed by Newman-Keuls test (intragroup analyses), by repeated measures ANOVA followed by Newman-Keuls test (intergroup analyses), and by two-tailed t-test (when applicable) (GraphPad Prism software, San Diego, CA). A 2-sided P value of less than 0.05 was considered to be statistically significant.
Results
In Sprague-Dawley rats, PNx led to hypertension, marked increases in plasma creatinine, development of hypertension, and oxidative stress as assessed by plasma levels of carbonylated protein (Table 1). Plasma levels of MBG in PNx rats were elevated 4-fold vs. that in sham-operated animals (Figure 1A). Rats subjected to PNx treated with vehicle developed cardiac hypertrophy (Figure 2A) which was accompanied by activation of cardiac oxidative stress assessed by carbonylated protein and DHE staining (Figure 2B–C). Development of renal failure in rats was also associated with cardiac fibrosis assessed by computer assisted morphological analysis and increased levels of collagen-1 in left ventricular myocardium (Figure 3) as we have previously reported.5,6 Cardiac levels of Fli-1 in PNx rats were markedly reduced vs. that in sham-operated animals (Figure 3A).
Plasma levels of MBG (A), creatinine (B), and creatinine clearance (C) in sham-operated (Sham) and 5/6 nephrectomized (PNx) rats, treated with vehicle (Veh), Digibind (DG) or anti-MBG mAb (3E9). Effects of administration of 3E9 anti-MBG mAb and of Digibind to PNx rats on systolic BP (D). Means ± SEM from 6 observations. A: (*) - P<0.01 vs. Sham by two-tailed t-test. B and C: (*) - P<0.05, (**) - P<0.001 vs. Sham, (#) - P<0.05, (##) - P<0.01 vs. vehicle by one-way ANOVA followed by Newman-Keuls test. D: By repeated measures ANOVA and Newman-Keuls test: Digibind vs. vehicle – P<0.05; 3E9 mAb vs. vehicle – P<0.01; Digibind vs. 3E9 – P<0.01.
Effects of administration of 3E9 anti-MBG mAb and of Digibind to PNx rats on heart weight (A), and on cardiac levels of oxidative stress assessed by measurement of carbonylated protein (B) and DHE fluorescence intensity (C; upper panels, representative measurements; lower panel – quantitative measurements, mean±SEM of 4 densiometry determinations). PNx – 5/6 nephrectomized rats. Vehicle – PNx rats administered vehicle. Means ± SEM from 6 observations. A–C: By one-way ANOVA and Newman-Keuls test (*) – P<0.05 and (**) – P<0.01 vs. Sham. (#) – P<0.05 and (##) – P<0.01 vs. vehicle-treated PNx rats.
Representative (upper panel) and quantitative (lower panel, mean±SEM of 4 densiometry measurements) analysis of Fli-1 (A) and collagen-1 (B) Western blots performed on cardiac tissues from the different groups. Actin was used to control loading. C – representative (upper panel) and quantitative (lower panel, mean±SEM of 4 densiometry measurements) trichrome-stained photomicrographs obtained from cardiac tissue derived from the different experimental groups. Sham – sham-operated rats, PNx – rats following 5/6 nephrectomy, Veh – PNx rats administered vehicle, DG – PNx rats administered Digibind, 3E9 – PNx rats administered 3E9 anti-MBG mAb. By one-way ANOVA and Newman-Keuls test: (*) – P<0.01 vs. Sham; (#) – P<0.05, (##) – P<0.01 vs. Veh.
Table 1
Physiological measurements in controls rats and after 5/6 nephrectomy
| Variable | Sham (n=8) | PNx (n=18) |
|---|---|---|
| Body weight (grams) | 453 ± 7 | 432 ± 10 |
| Plasma creatinine (mg/dL) | 0.35 ± 0.07 | 1.27 ± 0.09* |
| Creatinine clearance (ml/min) | 5.60 ± 0.55 | 0.84 ± 0.05* |
| Hematocrit (%) | 45±1 | 39±1* |
| Serum Na (mmol/L) | 141±1 | 143±2 |
| Serum K (mmol/L) | 5.6±0.3 | 6.3±0.3 |
| Systolic blood pressure (mmHg) | 110 ± 5 | 189 ± 14* |
| Heart rate (beats/min) | 375 ± 4 | 385 ± 3 |
| Heart weight/body weight, ×103 | 0.26 ± 0.03 | 0.36 ± 0.03* |
| Plasma carbonylated protein (pmol/mg protein) | 170 ± 10 | 470 ± 90* |
Physiological measurements in control rats and in the animals following 5/6 nephrectomy (PNx). Means ± SEM.
Figures 1 and and22 summarize data on the effect of administration of 3E9 anti-MBG mAb and Digibind on blood pressure, heart weight and cardiac levels of carbonylated protein. A single administration of Digibind produced a transient decrease in arterial pressure while, in contrast, administration of 3E9 mAb resulted in a substantial and sustained decrease in systolic blood pressure following 1 week of antibody administration (Figure 1D). In addition to depressor effect, administration of 3E9 mAb and Digibind to PNx rats was associated with reduction in the serum levels of creatinine and a concomitant increase in creatinine clearance (Figure 1B,C).
Administration of both antibodies was associated with a reduction in cardiac weight and a decrease in the cardiac expression of carbonylated protein (Figure 2), as well as increase in the left ventricular expression of Fli-1 protein (Figure 3A) along with reductions in cardiac collagen-1 protein expression and morphological evidence of fibrosis (Figure 3B,C). These effects of 3E9 mAb were more pronounced as compared to those of Digibind.
Discussion
The main observation of the present experiment is that a single administration of a mAb against an endogenous Na/K-ATPase inhibitor, MBG, to rats with experimental renal failure produced a sustained depressor effect associated with a dramatic reduction in cardiac fibrosis and increase in cardiac levels of Fli-1, a negative regulator of collagen synthesis. Fli-1 belongs to a family of Ets oncogenes, and it competes with another transcription factor, ETS-1, to maintain a balance between stimulation and repression of Col1a2 gene promoter.8 Fli-1 is implicated in dermal fibrosis and it exhibits direct effect on collagen-1 synthesis in dermal fibroblasts,8 and we have observed that decreases in Fli-1 expression appear to be necessary for CTS to stimulate fibroblast collagen production.7
Our present results demonstrate that blockade of the CTS-Na/K-ATPase signal cascade can actually reverse established cardiac fibrosis in the PNx model. Our previous studies demonstrated that cardiac fibrosis was well established after 4 weeks following PNx in the Sprague Dawley rat.5,6 In the current study, treatment was administered to rats at 4 weeks following PNx, and animals were sacrificed one week later. While the vehicle treatment group, studied 5 weeks after PNx, demonstrated similar degrees of cardiac hypertrophy and fibrosis to what we had previously reported at 4 weeks,5,6 both Digibind and 3E9 treated animals had remarkable clearing of myocardial fibrosis over the next week accompanied by up-regulation of cardiac Fli-1. These data further indicate that MBG-dependent Fli-1 downregulation is implicated in the pathogenesis of cardiac fibrosis seen with experimental chronic renal failure. Our present observations that immunoneutralization of MBG was accompanied by reduction in systemic and cardiac levels of oxidative stress agrees with previous data demonstrating that generation of reactive oxygen species is implicated in MBG-dependent cell signaling.18,19
Previous studies in PNx rats implemented remnant kidney fibrosis in the progression of renal failure in this model.20,21 Although absence of renal morphology data is a limitation of the present study, we found that in PNx rats immunoneutralization of CTS with both 3E9 mAb and Digibind reduced plasma creatinine concentration and produced a substantial increase in creatinine clearance. This observation suggests that in PNx rats beneficial effects of MBG immunoneutralization are not limited to pressor and cardiac effects, and warrants further studies of the role of CTS in pathogenesis of renal fibrosis.
Notably, in the present study, in the case of Digibind, reduction of cardiac fibrosis occurred in the absence of a sustained blood pressure lowering effect. Thus, in the present experiment, Digibind and 3E9 mAb in PNx rats exhibited comparable anti-fibrotic effects in the presence of markedly varying effects on the blood pressure within 1 week after a single injection; while the blood pressure-lowering effect of anti-MBG mAb was profound and sustained, the depressor effect of Digibind was minor and transient. We, therefore, propose that in the present study, both antibodies exhibited blood pressure-independent antifibrotic effects, which agrees with our previous data demonstrating that a pronounced anti-fibrotic effect of active immunization of PNx rats against MBG was associated with a very minor effect on the blood pressure.5,6
The 3E9 anti-MBG mAb employed in the current study is highly selective for bufadienolide CTS and does not cross react with cardenolide sodium pump inhibitors and other steroid hormones.10 Thus, in a DELFIA immunoassay, the 3E9 mAb exhibited substantial cross-reactivity only with two bufadienolides, telocinobufagin, a possible precursor of MBG which was reported to be elevated in plasma of patients with uremia,22 and cinobufotalin which differs from MBG in having one extra hydroxyl group.10 Previously we reported that following HPLC-fractionation of CTS from preeclamptic placentae, a competitive immunoassay based on Digibind exhibited reactivity to HPLC fractions having retention times similar to that seen with MBG and other bufadienolides, but no to ouabain-like immunoreactive material.23 Most recently, in patients with chronic kidney disease and in PNx rats we demonstrated that increase in plasma CTS detected by Digibind is likely to reflect an increase in the levels of MBG.24 These observations suggest that in renal failure MBG represents a target for Digibind.
In conclusion, in experimental chronic renal failure, elevated levels of MBG contribute to hypertension and induce cardiac fibrosis via suppression of Fli-1, representing a potential target for therapy. The effectiveness of 3E9 mAb for reversing the cardiac disease in PNx animals and the fact that 3E9 mAb exhibits a long-lasting effect following a single injection, suggests a potential role for MBG immunoneutralization in patients with uremic cardiomyopathy.
Acknowledgements
These studies were supported by Intramural Research Program, National Institute on Aging, NIH (OVF, and AYB) and by NIH grant HL071556 (JIS).