Author + information
- Received June 9, 2014
- Revision received August 14, 2014
- Accepted August 22, 2014
- Published online January 1, 2015.
- Laurent Calvier, PhD∗,
- Ernesto Martinez-Martinez, PhD†,‡,
- Maria Miana, PhD†,
- Victoria Cachofeiro, PhD†,
- Elodie Rousseau∗,
- J. Rafael Sádaba, MD‡,§,
- Faiez Zannad, PhD, MD∗,‖,¶,
- Patrick Rossignol, PhD, MD∗,‖,¶ and
- Natalia López-Andrés, PhD∗,‡∗ ()
- ∗INSERM, Université de Lorraine UMR 1116, Vandoeuvre-Lès-Nancy, France
- †Universidad Complutense de Madrid, Madrid, Spain
- ‡Navarrabiomed-Fundación Miguel Servet, Pamplona, Spain
- §Department of Cardiac Surgery, Complejo Hospitalario de Navarra, Pamplona, Spain
- ‖CHU Nancy, INSERM Clinical Investigation Center, CIC 9501, Vandoeuvre-Lès-Nancy, France
- ¶F-CRIN INI-CRCT (Cardiovascular and Renal Clinical Trialists), Nancy, France
- ↵∗Reprint requests and correspondence:
Dr. Natalia López-Andrés, Cardiovascular Translational Research, Navarrabiomed (Fundación Miguel Servet), Irunlarrea 3, 31008 Pamplona, Spain.
Objectives This study investigated whether galectin (Gal)-3 inhibition could block aldosterone-induced cardiac and renal fibrosis and improve cardiorenal dysfunction.
Background Aldosterone is involved in cardiac and renal fibrosis that is associated with the development of cardiorenal injury. However, the mechanisms of these interactions remain unclear. Gal-3, a β-galactoside–binding lectin, is increased in heart failure and kidney injury.
Methods Rats were treated with aldosterone-salt combined with spironolactone (a mineralocorticoid receptor antagonist) or modified citrus pectin (a Gal-3 inhibitor), for 3 weeks. Wild-type and Gal-3 knockout mice were treated with aldosterone for 3 weeks. Hemodynamic, cardiac, and renal parameters were analyzed.
Results Hypertensive aldosterone-salt–treated rats presented cardiac and renal hypertrophy (at morphometric, cellular, and molecular levels) and dysfunction. Cardiac and renal expressions of Gal-3 as well as levels of molecular markers attesting fibrosis were also augmented by aldosterone-salt treatment. Spironolactone or modified citrus pectin treatment reversed all of these effects. In wild-type mice, aldosterone did not alter blood pressure levels but increased cardiac and renal Gal-3 expression, fibrosis, and renal epithelial-mesenchymal transition. Gal-3 knockout mice were resistant to aldosterone effects.
Conclusions In experimental hyperaldosteronism, the increase in Gal-3 expression was associated with cardiac and renal fibrosis and dysfunction but was prevented by pharmacological inhibition (modified citrus pectin) or genetic disruption of Gal-3. These data suggest a key role for Gal-3 in cardiorenal remodeling and dysfunction induced by aldosterone. Gal-3 could be used as a new biotarget for specific pharmacological interventions.
Aldosterone (Aldo) is a well-known key regulator of blood pressure (BP) and electrolytic balance that acts classically via an intracellular mineralocorticoid receptor (MR) (1). A growing body of evidence suggests that Aldo plays an important pathophysiological role in cardiac, vascular, and renal remodeling via its MR by promoting oxidative stress, inflammation, fibrosis, and hypertrophy (2–4). The pivotal role of Aldo in cardiac and renal fibrosis is reinforced at the cellular level in tubule epithelial cells and in cardiac fibroblasts where Aldo increases collagen synthesis (5,6). However, the precise mechanism responsible for Aldo-induced cardiac and renal fibrosis remains to be determined, and despite advances in treatment of both cardiovascular and kidney diseases, cardiorenal injury remains a major clinical problem (7).
Galectin-3 (Gal-3) is a protein member of a β-galactoside binding lectin family, localized in the nucleus, cytoplasm, cell surface, and extracellular space (8). Several inhibitors of Gal-3 have been described, such as the modified citrus pectin (MCP) (9), a complex water-soluble indigestible polysaccharide rich in β-galactose. The expression of this lectin has been reported in many tissues (10) (including heart and kidney) and cells (in fibroblasts , endothelial cells , epithelial cells of tubules or collecting ducts , and inflammatory cells ). This lectin induces cardiac and renal fibrosis that leads to cardiac dysfunction (11) or acute kidney injury (13) in different experimental models of heart failure (HF) and kidney injury. Our group recently showed that Gal-3 is up-regulated by Aldo and that it mediates the inflammatory and fibrotic response to Aldo in vascular smooth muscle cells both in vitro and in vivo, indicating a key role for this lectin in Aldo-induced vascular fibrosis (15). In humans, the serum Gal-3 level has been correlated with serum markers of cardiac extracellular matrix turnover and therefore Gal-3 emerges as a biomarker associated with HF onset, morbidity, and mortality (16). In addition to this cardiac association, it has been shown that plasma Gal-3 is associated with renal impairment in patients with or without HF (17). The predictive value of Gal-3 appeared to be stronger in patients with HF with preserved ejection fraction, postulating that Gal-3 might be a particularly useful biomarker in HF with preserved ejection fraction (18). However, although previous studies have investigated separately the effects of Aldo and Gal-3 on cardiac remodeling and function, the interaction between these 2 factors and the pharmacological blockade or gene inactivation of Gal-3 have never been explored in the heart and the kidney in the context of high Aldo levels.
The hypothesis that we explore here is that Gal-3 is involved in Aldo-induced cardiac and renal fibrosis and dysfunction, and, therefore, Gal-3 could be a new key factor in the development of the cardiac and renal injuries. The present study was designed to examine the protective effect of the Gal-3 inhibition or absence in the progression of the cardiac and renal injuries using 2 animal models: 1) hypertensive hyperaldosteronism model of rats (treated with Aldo-salt) in combination with the Gal-3 inhibitor MCP; and 2) normotensive model of wild-type (WT) and Gal-3 knockout (KO) mice infused with Aldo with a normal salt diet.
Detailed materials and methods are available in the Online Appendix.
The investigation was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
For the Gal-3 inhibition model, adult male Wistar rats were treated for 3 weeks with vehicle (n = 10), Aldo-salt (1 mg/kg/day diluted in sunflower oil and administered by subcutaneous injection and 1% NaCl as drinking water, n = 10), Aldo-salt plus spironolactone (200 mg/kg/day, n = 10), Aldo-salt plus MCP (100 mg/kg/day, n = 9), spironolactone (n = 7), or MCP (n = 5) alone. After 2 weeks of treatment, urine was collected in metabolic cages. At the end of the treatment, hemodynamic parameters were evaluated.
For the Gal-3 ablation model, adult male C57BJ6 WT mice and Gal-3 KO mice (19) were infused for 3 weeks with Aldo (1 mg/kg/day, osmotic minipump) or vehicle (n = 7, each group). Tail cuff blood pressure was monitored throughout the treatment.
For histology, sections were stained with Sirius red. For immunohistochemistry, sections were incubated with primary antibody, then with peroxidase-labeled secondary antibody and revealed using a 3,3′-diaminobenzidine substrate kit (Vector Laboratories, Burlingame, California). Classical electrophoresis was performed on sodium dodecyl sulfate polyacrylamide gels and transferred to a nitrocellulose membrane. Reverse-transcription polymerase chain reaction was performed with SYBR green polymerase chain reaction technology. Protein concentrations were measured by an enzyme-linked immunosorbent assay according to the manufacturer's instructions. Urinary Na+ and K+ concentrations were measured by flame photometry (IL943, Instrumentation Laboratory, Bedford, Massachusetts).
Because means were found very close from the medians, results are presented as mean ± SEM, computed from the average measurements obtained from each group of animals. Normal distribution of data was checked by means of the Shapiro-Wilk test, and a Levene statistic test was performed to check the homogeneity of variances. Differences among more than 2 experimental conditions were tested by the analysis of variance 1-way test, followed by the Scheffé test to analyze differences between groups. The unpaired Student t test or the Mann-Whitney U test was used to assess statistical differences between 2 experimental conditions. Values of p < 0.05 were considered significant. With 80% power and 5% alpha error rate, a sample size of 10 subjects was needed to observe a significant 50% difference between the histological scores of the 2 groups. Because the study was exploratory, no adjustment for multiple testing was made; the relevance of significant results was appreciated in the light of other findings.
Inhibition or lack of Gal-3 blocks aldo-induced hypertension, cardiac remodeling, fibrosis, and dysfunction
Aldo-salt treatment induced (p < 0.05) an increase in systolic BP and diastolic BP as well as in pulse pressure (Table 1). All the hemodynamic effects were prevented (p < 0.05) by cotreatment with spironolactone or MCP. At the cardiac level, Aldo-salt treated rats presented an increase (p < 0.05) in heart weight to body weight ratio, heart rate, left ventricular (LV) end-diastolic pressure, and first derivative of the LV pressure decrease over time. Cardiac dysfunction was accompanied by an increase (p < 0.05) in LV wall thickness and LV cross-sectional area in Aldo-salt-treated rats. Both cardiac dysfunction and hypertrophy were prevented (p < 0.05) by the MR antagonist spironolactone or the Gal-3 inhibitor MCP cotreatment. At the cellular level, myocardium from Aldo-salt–treated rats presented a significant increase in cardiomyocyte transverse diameter (22%, p < 0.05), cross-sectional area (48%, p < 0.05) and length (11%, p < 0.05) (Table 1). Cotreatment with spironolactone or MCP totally blocked (p < 0.05) the Aldo-salt–induced cardiomyocyte length increase (Table 1).
Hypertesive Aldo-salt–treated rats presented increased cardiac Gal-3 expression at both the mRNA (Figure 1A) and protein (Figure 1B) levels. Cotreatment with spironolactone or MCP abolished (p < 0.05) Aldo-salt–induced cardiac Gal-3 up-regulation at the mRNA (Figure 1A) and protein (Figure 1B) levels.
Aldo-salt–enhanced mRNA and protein synthesis of collagen type I and the profibrotic marker transforming growth factor-β were inhibited by either spironolactone or MCP cotreatment (p < 0.05) (Figures 1C and 1D). Histological analyses revealed that Aldo-salt treatment increased myocardial interstitial (150%, p < 0.05) and perivascular (65%, p < 0.05) collagen deposition (Figure 1E) compared with controls. These increases were blocked (p < 0.05) by spironolactone or MCP cotreatment.
Neither spironolactone nor MCP treatment affected all measured parameters in the absence of Aldo (Online Table 1).
To complete the understanding of Gal-3 actions in Aldo-induced cardiac fibrosis and dysfunction, WT and Gal-3 KO mice were challenged with Aldo only to avoid an increase in BP and its potential confounding effects. At baseline, no differences in general, hemodynamic, and cardiac parameters were observed among the 2 strains of mice (Table 2). After Aldo treatment, no difference in body weight, heart weight/body weight, or systolic BP were noticed in WT or KO mice (Table 2). However, Aldo-treated WT mice presented increased cardiac Gal-3 expression at mRNA and protein levels (Figures 1F and 1G). Moreover, Aldo treatment increased cardiac collagen type I (22%, p < 0.05) and type III (46%, p < 0.05) protein expressions (Figure 1H) in WT mice, whereas KO mice were specifically resistant to Aldo-induced collagen type I increase (p < 0.05). Aldo induced interstitial and perivascular collagen deposition in WT mice, whereas KO mice were resistant, as shown in the representative photographs stained for collagen (Figure 1I).
Inhibition or lack of Gal-3 blocks aldo-induced alterations in renal tubular cells and renal fibrosis
Aldo-salt treatment induced (p < 0.05) renal hypertrophy (increased kidney weight to body weight ratio) and glomerular hypertrophy, increased glomerular filtration rate (assessed by the creatinine clearance), increased urine sodium to potassium ratio, and increased albuminuria and urine neutrophil gelatinase-associated lipocalin (NGAL), the latter indicating tubular lesions (Table 1). Renal and glomerular hypertrophy, hyperfiltration, albuminuria, and tubular lesions were prevented (p < 0.05) by spironolactone cotreatment, whereas MCP cotreatment failed to prevent kidney hypertrophy, glomerular hypertrophy, and glomerular hyperfiltration. However, MCP treatment protected the kidney from the increase in urine sodium to potassium ratio, albuminuria and urine NGAL (Table 1).
Hypertensive Aldo-salt–treated rats presented increased renal Gal-3 expression at both mRNA (Figure 1A) and protein (Figure 1B) levels. These increases were abolished (p < 0.05) by spironolactone or MCP cotreatments. Renal Gal-3 expression was located in proximal and distal tubules (epithelial cells) but not in the glomeruli (Online Figure 1).
Spironolactone or MCP cotreatment inhibited (p < 0.05) Aldo-salt treatment–induced mRNA and protein synthesis of collagen type I and transforming growth factor-β in the kidney (Figures 2C and 2D). This fibrotic response was confirmed by the complementary histological analysis. Aldo-salt–treated rats presented increased interstitial (141%, p < 0.05), glomerular (39%, p < 0.05), and perivascular (79%, p < 0.05) collagen compared with controls (Figure 2E). Cotreatment with spironolactone inhibited these collagen increases. MCP cotreatment blocked Aldo-salt induced interstitial and perivascular collagen depositions but not glomerular collagen deposition.
Neither spironolactone nor MCP treatment affected all measured parameters in the absence of Aldo (Online Table 2).
In WT and Gal-3 KO mice, no difference in plasma concentration of albumin, creatinine, and NGAL were noted after Aldo treatment (Table 2). However, Aldo treatment induced (p < 0.05) an increase in kidney weight to body weight ratio and glomerular hypertrophy in both WT and Gal-3 KO mice (Table 2). Normotensive Aldo-treated WT mice presented increased renal Gal-3 expression at mRNA and protein levels (Figures 2F and 2G). Aldo treatment increased renal collagen type I (46%, p < 0.05) protein expression in WT mice (Figure 2H). Gal-3 KO mice were specifically resistant to Aldo-induced interstitial and perivascular collagen deposition compared with WT mice. However, the lack of Gal-3 failed to reverse Aldo-induced glomerular collagen deposition, as illustrated by the collagen staining (Figure 2I).
Inhibition or lack of Gal-3 blocks aldo-induced epithelial-mesenchymal transition
The kidney of Aldo-salt–treated rats presented a decrease in β-catenin and E-cadherin as well as an increase in fibronectin and α-smooth muscle actin (α-SMA) mRNA (Figure 3A) and protein expressions in tubules (Figure 3B) compared with controls. These changes were blocked (p < 0.05) by spironolactone or MCP cotreatment. These results were confirmed by histological analyses (Figure 3C).
Neither spironolactone nor MCP treatment affected all measured parameters in the absence of Aldo (Online Table 2).
In the renal tubules of the mice model, Aldo treatment decreased β-catenin (38%, p < 0.05) and increased α-SMA (35%, p < 0.05) protein expression in WT mice (Figure 3D). Gal-3 KO mice were resistant to an Aldo-induced decrease in β-catenin and an increase in α-SMA expression (p < 0.05). These results were confirmed by histological analyses (Figure 3E).
We have demonstrated here the role of Gal-3 in cardiac and renal fibrosis induced by Aldo and the protective effect of the Gal-3 inhibitor MCP or the constitutive absence of Gal-3 in the progression of the cardiac and renal injuries.
Gal-3 is involved in heart (11) and kidney (13) diseases associated with fibrosis, leading to organ remodeling, dysfunction, and HF. However, the precise contribution of Gal-3 in HF, particularly with preserved ejection fraction, is unclear. From the pathophysiology of HF with preserved ejection fraction (20), which is characterized by hypertrophy, matrix apposition, and myocardial stiffening, it is only natural that a matrix and fibrosis marker like Gal-3 may play an important role.
In the heart, Aldo-salt induces MR-dependent cardiomyocyte hypertrophy, cardiac fibrosis, and subsequently LV dilation and increase in LV weight. Functionally, these changes result in LV diastolic dysfunction (2). We demonstrate here for the first time that the Gal-3 inhibitor MCP prevents all these Aldo-salt–induced cardiac adverse effects. It has been reported that genetic disruption of Gal-3 or its inhibition attenuated the progression of cardiac fibrosis, remodeling, and dysfunction in angiotensin II–treated animals (21). These results confirm previous observations on the protective effects of Gal-3 blockade in the aorta (15) and strengthen the potential of Gal-3–targeted therapies in the treatment of HF (21).
In the kidney, Aldo-salt–treated rats present an MR-dependent epithelial-mesenchymal transition and increased interstitial, glomerular, and vascular fibrosis. Aldo-salt treatment induced kidney hypertrophy, glomerular hypertrophy, hyperfiltration, albuminuria as well as increased sodium and NGAL urine excretion, according to previously published data (6). Interestingly, MCP is protective in experimental acute nephropathy with modulation of fibrosis. Here, we complete the understanding of the MCP protective actions demonstrating that MCP prevented tubular damage specifically but not glomerular damage. This is consistent with the fact that Gal-3 expression is specifically located in tubules and not in glomeruli. These data suggest that MCP acts via Gal-3 inhibition only at the tubular level.
Taken together, our observations indicate that MCP, via its blocking action on Gal-3, can protect against Aldo-induced cardiac and renal fibrosis and dysfunction. That MCP alone does not alter BP level but blunts Aldo-induced hypertension is an exciting finding that warrants further dedicated studies, especially at the levels of tubular function and vascular properties. Indeed, we have already reported that MCP blocked Aldo-salt–induced aortic stiffness (15), which could be one of several parameters acting on BP regulation. In Aldo-salt–treated rats, the hypertension is mainly due to the salt supplement. Therefore, to exclude hypertension, which is a confounding factor, and to exclude non–Gal-3–dependent effects of any MCP side-effect treatment, we used a WT and Gal-3 KO mice model challenged with Aldo without salt and thus without BP variation. Regarding cardiac and renal function, it has been reported that there is no baseline difference between Gal-3 KO and WT mice (15,21,22). Our present mouse model demonstrated that the lack of Gal-3 protected against Aldo-induced cardiac and tubular fibrosis and the renal epithelial-mesenchymal transition and failed to protect against glomerular fibrosis and hypertrophy. These data reinforce the critical role of Gal-3 in Aldo-induced early cardiac and renal remodeling, independent of BP variation.
One limitation of this study is the use of experimental approaches that are not physiological. To demonstrate these results, we used 2 experimental animal models of hyperaldosteronism, in which it has been reported that Aldo affects the cardiovascular and renal systems, inducing LV fibrosis, impairing diastolic LV filling, and promoting hyperfiltration (2,3). These experimental models (rat and mice) are completely dependent on Aldo. However, these results open the field for further studies to complete our understanding of the pathways and mechanisms revealed here.
Long-term Aldo exposure induced heart and kidney remodeling, which may ultimately lead to HF and kidney insufficiency, one aggravating the other in a vicious cycle. We propose that Gal-3 is a key player and a potential therapeutic target in cardiac and renal fibrosis induced by Aldo, both of which are major contributors to cardiorenal injury. The dietary supplement MCP, a Gal-3 inhibitor, represents in this context a great opportunity and could be used in patients with cardiorenal injuries related to Aldo increase. More in-depth mechanistic studies would be needed to study how MCP could block Aldo hypertensive effects. Further clinical studies are required to establish the potential therapeutic benefit of Gal-3 inhibition with MCP in patients with cardiorenal diseases.
The authors thank Raquel Jurado and El moghrabi Soumaya for technical help, Prof. Simon Thornton for editing help, and Renaud Fay for statistical analysis help.
For detailed materials and methods as well as supplemental tables and a figure, please see the online version of this article.
Funded by INSERM, Programme National de Recherche Cardiovasculaire, Région Lorraine; the 6th EU-FP Network of Excellence Ingenious HyperCare (contract LSHM-CT-2006–037093); the FP7 HOMAGE (grant agreement FP7 305507) and FIBRO-TARGETS project (grant agreement FP7 602904); Fondo de Investigaciones Sanitarias (PI12/01729); Red de Investigación Cardiovascular (RD12/0042/0033); BG Medicine; and Miguel Servet (contract CP13/00221). BG Medicine has certain rights with respect to the use of Gal-3 in heart failure. Dr. Rossignol has received a research grant from BG Medicine. Drs. Zannad and López-Andrés have received honoraria and grants from BG Medicine. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- blood pressure
- heart failure
- left ventricular
- modified citrus pectin
- mineralocorticoid receptor
- neutrophil gelatinase associated lipocalin
- smooth muscle actin
- Received June 9, 2014.
- Revision received August 14, 2014.
- Accepted August 22, 2014.
- American College of Cardiology Foundation
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