Telmisartan ameliorates cardiac fibrosis and diastolic function in cardiorenal heart failure with preserved ejection fraction

Abstract

Chronic kidney disease (CKD) is a major contributor to the development of heart failure with preserved ejection fraction (HFpEF), whereas the underlying mechanism of cardiorenal HFpEF is still elusive. The aim of this study was to investigate the role of cardiac fibrosis in a rat model of cardiorenal HFpEF and explore whether treatment with Telmisartan, an inhibitor of renin-angiotensin-aldosterone system (RAAS), can ameliorate cardiac fibrosis and preserve diastolic function in cardiorenal HFpEF. Male rats were subjected to 5/6 subtotal nephrectomy (SNX) or sham operation (Sham), and rats were allowed four weeks to recover and form a stable condition of CKD. Telmisartan or vehicle was then administered p.o. (8 mg/kg/d) for 12 weeks. Blood pressure, brain natriuretic peptide (BNP), echocardiography, and cardiac magnetic resonance imaging were acquired to evaluate cardiac structural and functional alterations. Histopathological staining, real-time polymerase chain reaction (PCR) and western blot were performed to evaluate cardiac remodeling. SNX rats showed an HFpEF phenotype with increased BNP, decreased early to late diastolic transmitral flow velocity (E/A) ratio, increased left ventricular (LV) hypertrophy and preserved ejection fraction (EF). Pathology revealed increased cardiac fibrosis in cardiorenal HFpEF rats compared with the Sham group, while chronic treatment with Telmisartan significantly decreased cardiac fibrosis, accompanied by reduced markers of fibrosis (collagen I and collagen III) and profibrotic cytokines (α-smooth muscle actin, transforming growth factor-β1, and connective tissue growth factor). In addition, myocardial inflammation was decreased after Telmisartan treatment, which was in a linear correlation with cardiac fibrosis. Telmisartan also reversed LV hypertrophy and E/A ratio, indicating that Telmisartan can improve LV remodeling and diastolic function in cardiorenal HFpEF. In conclusion, cardiac fibrosis is central to the pathology of cardiorenal HFpEF, and RAAS modulation with Telmisartan is capable of alleviating cardiac fibrosis and preserving diastolic dysfunction in this rat model.

Keywords: Heart failure with preserved ejection fraction; cardiac fibrosis; cardiorenal syndrome; chronic kidney disease; diastolic function; renin-angiotensin-aldosterone system.

Figure 1.

Flowchart of timeline and number of rats used in each group in applied study design. 5/6 subtotal nephrectomy (SNX) was performed in rats to induce initial kidney injury by first resecting the whole right kidney and subsequently resecting two-thirds of the left kidney one week later (0 w, which is right after the whole surgery). A group of sham-operated rats was used as control (Sham). After the surgery, all rats were allowed four weeks to recover and form a stable condition of chronic kidney disease. Then, the SNX rats were randomly divided into two groups and intragastrically administered vehicle (SNX+Veh), or Telmisartan (SNX + Tel) for an additional 12 weeks. Blood and urine collections were performed at Baseline (before the initial surgery, which is one week before 0 w) and on week 4, 8, 12, and 16 after the whole surgery to evaluate renal function. Echocardiography and blood pressure were measured at Baseline before the initial surgery and at week 16 after the whole SNX surgery. Cardiac MRI was conducted at week 16. Then, the rats were euthanized, and renal/cardiac pathological examination, real-time PCR, and western blot analysis were performed at week 16.

Figure 2.

Chronic renal injury in cardiorenal HFpEF and alleviated renal injury after treatment with Telmisartan. The mean ± SD of serum creatinine (a), blood urea nitrogen (b), and 24 h urine protein excretion (c) from Baseline to week 16. (d) Histopathological staining with anti-CD68 and calculated percentages of macrophages in renal sections. (e) Masson staining and calculated percentages of fibrosis in renal sections. Scale bar in (e), 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001 for SNX+Veh vs. Sham; #P < 0.05, ##P < 0.01, ###P < 0.001 for SNX+Veh vs. SNX+Tel. Masson indicates Masson trichrome. (A color version of this figure is available in the online journal.)

Figure 3.

Cardiac function assessment in rats with cardiorenal HFpEF and effects of chronic treatment with Telmisartan on cardiorenal HFpEF phenotype. The mean ± SD of systolic blood pressure (a), diastolic blood pressure (b), and BNP (c) at Baseline and at week 16 after the surgery. The E/A ratio in echocardiography at week 16 (d) and the quantified mean ± SD of E/A ratio (e), and ejection fraction (f) at Baseline and at week 16 after the surgery. BNP indicates brain natriuretic peptide; E/A: early to late diastolic transmitral flow velocity. (A color version of this figure is available in the online journal.)

Figure 4.

Left ventricular hypertrophy measurement by cardiac MRI and histology. (a) Cardiac Cine MRI of the same slice of the heart at end-diastole (top row) and end-systole (bottom row) of the three groups at week 16 and (b) the mean ± SD of IVSd and IVSs as well as (c) the mean ± SD of LVEDV and LVESV at week 16 after the surgery. (d) Cardiac H&E staining of myocytes in cross-section at week 16 and the calculated mean ± SD of myocyte cross-sectional diameter. (e) The mean ± SD of LV mass at week 16. Scale bar in (a), 2.5 mm. Scale bar in (d), 50 μm. MRI indicates magnetic resonance imaging; IVSd: interventricular septal thickness at end-diastole; IVSs: interventricular septal thickness at end-systole; LVEDV: left ventricular end-diastolic volume; LVESV: left ventricular end-systolic volume; H&E: hematoxylin & eosin; LV: left ventricular. (A color version of this figure is available in the online journal.)

Figure 5.

Characterization of cardiac fibrosis in cardiorenal HFpEF and reduced cardiac fibrosis and collagen deposition after treatment with Telmisartan. (a) Masson staining and (b) the calculated mean ± SD of percentage of cardiac fibrosis at week 16. (c) Real-time PCR and (d) western blotting of collagen I and collagen III in heart tissues at week 16 and the calculated mean ± SD of protein/β-actin ratios of collagen I and collagen III. Scale bar in (a), 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001 for SNX+Veh vs. Sham; #P < 0.05, ##P < 0.01, ###P < 0.001 for SNX+Veh vs. SNX+Tel. (A color version of this figure is available in the online journal.)

Figure 6.

Characterization of cardiac profibrotic cytokines expression in cardiorenal HFpEF. (a) Immunohistochemical staining of α-SMA, TGF-β1, and CTGF at week 16. (b) Real-time PCR and (c) western blot of α-SMA, TGF-β1, and CTGF in heart tissues and the calculated mean ± SD of protein/β-actin ratios. Scale bar in (a), 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001 for SNX+Veh vs. Sham; #P < 0.05, ##P < 0.01, ###P < 0.001 for SNX+Veh vs. SNX+Tel. (A color version of this figure is available in the online journal.)

Figure 7.

Reduced cardiac inflammation after treatment with Telmisartan and correlation analysis of cardiac inflammation and fibrosis. (a) Cardiac H&E staining and (b) CD68 immunohistochemical staining at week 16 and (c) the calculated mean ± SD of percentage of cardiac macrophages at week 16. (d) Cardiac inflammatory cells (H&E, left), macrophages (CD68, middle), and fibrosis (Masson, right) in adjacent cardiac slices and (e) the quantitative correlation analysis between percentage of macrophages area and percentage of fibrosis area (R² = 0.68; P < 0.001). Scale bar in (b) and (d), 100 μm. H&E indicates hematoxylin & eosin; Masson: Masson trichrome. (A color version of this figure is available in the online journal.)

Figure 8.

Immunohistological staining of macrophages and myofibroblasts in adjacent cardiac slices. Cardiac tissues of the SNX+Veh rats at week 16 were stained sequentially with anti-CD68 and α-SMA in adjacent slices. Although CD68-positive macrophages and α-SMA-positive myofibroblasts coexisted in the same clusters, they did not overlap. Scale bar, 100 μm. (A color version of this figure is available in the online journal.)