Hydrochlorothiazide Reduces Cardiac Hypertrophy,Fibrosis and Rho-Kinase Activation in DOCA-Salt Induced Hypertension

Abstract

Background:

Thiazides are one of the most common antihypertensive drugs used for hypertension treatment and hydrochlorothiazide (HCTZ) is the most frequently used diuretic for hypertension treatment. The Rho/Rho-kinase (ROCK) path plays a key function in cardiovascular remodeling. We hypothesized that in preclinical hypertension HCTZ reduces myocardial ROCK activation and consequent myocardial remodeling.

Methods:

The preclinical model of deoxycorticosterone (DOCA)-salt hypertension was used (Sprague–Dawley male rats). After 3 weeks, in 3 different groups: HCTZ, the ROCK inhibitor fasudil or spironolactone was added (3 weeks). After 6 weeks myocardial hypertrophy and fibrosis, cardiac levels of profibrotic proteins, mRNA levels (RT PCR) of pro remodeling and pro oxidative molecules and ROCK activity were determined.

Results:

Blood pressure, myocardial hypertrophy and fibrosis were reduced significantly by HCTZ, fasudil and spironolactone. In the heart, increased levels of the pro-fibrotic proteins Col-I, Col-III and TGF-β1 and gene expression of pro-remodeling molecules TGF-β1, CTGF, MCP-1 and PAI-1 and the pro-oxidative molecules gp91phox and p22phox were significantly reduced by HCTZ, fasudil and spironolactone. ROCK activity in the myocardium was increased by 54% (P < 0.05) as related to the sham group and HCTZ, spironolactone and fasudil, reduced ROCK activation to control levels.

Conclusions:

HCTZ reduced pathologic LVH by controlling blood pressure, hypertrophy and myocardial fibrosis and by decreasing myocardial ROCK activation, expression of pro remodeling, pro fibrotic and pro oxidative genes. In hypertension, the observed effects of HCTZ on the myocardium might explain preventive outcomes of thiazides in hypertension, specifically on LVH regression and incident heart failure.

Keywords

hydrochlorothiazide hypertrophy Rho-kinase cardiac remodeling hypertension

Introduction

At the present time global prevalence of human hypertension estimated conventionally is 31.1%^1,2 and possibly it will increase in the next years.² Hypertension is the major not inevitable risk factor for cardiovascular disease and all-cause global mortality.^3,4 At the same time, left ventricular (LV) remodeling is the major factor in hypertensive-induced target organ damage that involves LV mechanical and functional changes. Hypertensive LV hypertrophy (LVH) involves molecular and biochemical changes, myocardial interstitial and perivascular fibrosis and inflammatory infiltrates as well.^5
-7 LVH is found in ≈40% of patients with hypertension and it is an independent predictor of cardiovascular events and death.^8

-11 Since LVH prevention/regression by antihypertensive drugs is significantly associated to fewer cardiovascular events, it is most relevant to prevent and revert it.^12,13

Diuretics (mainly thiazides) are one of the most frequently used antihypertensive drugs to control blood pressure and currently they are recommended for starting antihypertensive treatment alone or as a combination.^14,15 Clinical studies in hypertension such as the ALLHAT study and more recently the LEGEND-HTN study confirm that cardiovascular as well as renal risk is significantly reduced in hypertensive patients treated with thiazides or thiazide-like diuretics.^16,17

Currently, hydrochlorothiazide (HCTZ) is one of the most regularly used diuretics for the treatment of hypertension by acting on the distal convoluted tubules and inhibiting the sodium chloride co-transporter system which leads to its diuretic effect as well as loss of potassium in the urine.^18,19 Initially, thiazides decrease extracellular volume and cardiac output, but the extracellular volume gradually returns to near normal levels in the long time. Apparently in this later step thiazides decrease peripheral resistance by a mechanism that involves reduction in vascular reactivity mediated by activation of vascular K+ channels,²⁰ vasorelaxation mediated by large-conductance Ca2+-activated K+) channels,^21,22 endothelium-dependent relaxing and nitric oxide release and also by inhibition of the RhoA-Rho kinase pathway.^23

-26 Nevertheless, the mechanism underlying the cardioprotective effects of HCTZ is not totally clear.

Rho/Rho-kinase (ROCK) plays a main role in the development of cardiovascular disease and remodeling by promoting endothelial cell barrier dysfunction, reactive oxygen species production, and inflammation in endothelial cells, contraction, migration, and proliferation in vascular smooth muscle cells, and transformation of fibroblasts to myofibroblast, proliferation of fibroblasts, and extracellular matrix synthesis in fibroblasts.²⁷ In cardiovascular remodeling due to hypertension the consequences of increased ROCK activity are through regulation of various cellular functions involving vascular smooth muscle cells, the cytoskeleton, motility and adhesion as well as expression of genes involved in cardiovascular and renal remodeling.²⁸ Enhanced ROCK activity has been observed in hypertensive patients an also in preclinical hypertension.^26,29

-33 Furthermore, ROCK inhibitors decrease BP in different hypertensive models.^26,34
-36 On the other side ROCK activation induces cardiomyocyte apoptosis, myocardial fibrosis, and myocardial hypertrophy.^37
-39

However, the role of HCTZ on hypertensive cardiac hypertrophy, remodeling and ROCK activation in the myocardium has not been previously assessed. We hypothesized here that in addition to lowering blood pressure, HCTZ reduces myocardial ROCK activation and consequent myocardial remodeling in preclinical hypertension.

Methods

The current preclinical study was formerly assessed and approved by the Committee of Research from the Medical School, Pontificia Universidad Católica de Chile. The experimental hypertensive model of deoxycorticosterone acetate (DOCA)-salt was here utilized in male rats (Sprague–Dawley, 150 ± 10 g, aged 5-6 weeks as formerly described.²⁶ A careful left nephrectomy was performed under intraperitoneal anesthesia using ketamine HCl (35 mg/kg) and xylazine (7 mg/kg). Then, DOCA (100 mg/kg subcutaneous; Steraloids Inc., Newport, Rhode Island, USA) was administered starting immediately after surgery, once per week. The animals were given drinking water containing sodium chloride (1%) and potassium chloride (0.4%).²⁶ Controls were sham operated rats. Three weeks after DOCA administration 5 hypertensive DOCA-salt groups were randomized during 3 additional weeks. One hypertensive group did not receive any antihypertensive drug during the whole 6 weeks and 4 additional hypertensive groups were randomized to the specific ROCK inhibitor fasudil (FAS, 50 mg/kg/day), or HCTZ (6 mg/kg/day), or spironolactone (Spiro, 100 mg/kg/day) by gavage for 3 more weeks. Spironolactone was used as a comparator with fasudil and HCTZ because of its well proved antihypertensive and anti-myocardial remodeling properties.⁴⁰ In human hypertension, spironolactone is mainly indicated for resistant hypertension.⁴¹ The sixth experimental group received a combination of fasudil (25 mg/kg/day) + HCTZ (4 mg/kg/day) by gavage for 3 weeks. Thus, 6 experimental groups were compared after 6 weeks.

The tail-cuff method was used once a week to measure systolic blood pressure (SBP). Six weeks after surgery, the 5 experimental groups were euthanized by using intraperitoneal anesthesia (ketamine HCl, 35 mg/kg and xylazine 7 mg/kg) and cervical dislocation. The heart was removed, carefully washed with saline buffer, one part of left ventricle was fixed with Bouin for 12 h and included in paraffin. The other part from left ventricle was homogenized as described²⁶ in cold lysis buffer containing Tris HCl, NP40, sodium chloride, sodium deoxycholate, ethylenediaminetetraacetic acid, aprotinin, leupeptin and phenylmethylsulphonyl fluoride. Samples were centrifuged (4 °C) and the content of proteins in the supernatants were measured with the Bradford assay. Afterward, the soluble fractions were used for Western blotting.

Histological and Morphometric Analysis

Relative cardiac weight (RCW, g/cm), was calculated as the ratio of cardiac weight (g) to the length of the tibia (cm).

Cross sections of the hearts were fixed in Bouin solution, embedded in paraffin. 5-µm tissue sections were stained with hematoxylin-eosin to be analyzed under light microscopy (Nikon Eclipse E400). The images were captured with a Nikon DS Fi1 camera and projected onto a monitor for analysis (NIS-Element BR software). LV area, posterior wall thickness (PWT) and anterior wall thickness (AWT) were measured. To determine PWT and AWT, 10 measurements of each LV were averaged. For measuring cardiomyocyte cross-sectional (μm²) and perimeter (μm), at least 100 cells in each animal were analyzed.

To assess interstitial fibrosis, 5-µm thick cross sections from the middle portion of the heart were stained with picrosirius red (Sirius Red 0.1% in acid picric saturated), to be examined under light microscopy (Nikon Eclipse E400 equipment). To determine myocardial fibrosis, the LV was divided into 2 areas for analysis: myocardium and sub-endocardium. For measuring myocardial collagen at least 32 images were analyzed of the area per study animal. Similarly, for determine the percentage of subendocardial collagen is analyzed at least 20 images of the area for each animal the percentage of total collagen was determined by adding the % myocardial collagen + % subendocardial collagen.

Collagen was additionally visualized under polarized light and quantified as freshly deposited collagen (green) and chronically deposited collagen (red). To determine freshly deposited collagen (green) and chronically deposited collagen (red), sirius red staining was imaged under polarized light. In order to quantify myocardial collagen under polarized light, captured color images were binarized to black and white by using the Fiji software, an open source image processing package based on ImageJ2 (adapted from Morais et al).⁴² Birefringent areas, corresponding to positive staining, were converted to black. Thus, white areas were excluded and the remaining black area was quantified in relation to the captured field area.

Western Blot Analysis

In order to determine ROCK activity in the myocardium, the ratio of phosphorylated to total myosin phosphatase target protein-1 (phospho-MYPT1) was measured by Western blot.²⁵ Besides, the protein cardiac levels of Collagen I, Collagen III and TGF-β1, were also assessed by Western blot as previously described.²⁵ In brief, the myocardial extracts were homogenized and separated by SDS-PAGE in 6% polyacrylamide gels, and electro transferred to nitrocellulose membranes. Membranes were blocked and incubated first with a primary antibody, Antiphospho-thr853-MYPT1 (CYP1025 CycLex Co., Ltd), anti-MYPT1 (BD Transduction Laboratories), anti-TGF-β1 (SC-146 Santa Cruz Biotecnology), anti-Collagen I (Col-I; AB749 Chemicon), Collagen III (Col-III; Biodesign International T40233 R), anti-β-Actin (Sigma A5316) overnight at 4°C. Subsequently to the washing, blots were incubated with a second antibody and later on with a tertiary antibody containing horseradish peroxidase. For assessing specific binding enhanced chemiluminescence with exposure to a Kodak film was used. Blots were quantified by scanning densitometry (Un-Scan-It software).

RT-PCR

The Trizol reagent was utilized to isolate total RNA (treated with DNAase) from the myocardium (1.5 μg). RNA was quantified with ultraviolet spectroscopy. Cardiac gene expression levels of the following genes was measured: Plasminogen activating inhibitor 1 (PAI-1), Monocyte Chemoattractant Protein-1 (MCP-1), TGF-β1, connective tissue growth factor (CTGF), gp91 phox and p22 phox through the transcription polymerase chain reaction (RT-PCR) technique. The specific primer sequences and amplification protocols were previously described 26. Following the PCR, the amplificated products were fractionated using a 1.5% agarose gel. The PCR products were observed under ethidium bromide stain and their optical density was measured by densitometry (computerized). The 18 S RNA levels were employed for normalization of the optical density of the RNA strips.²⁶

Statistical Analysis

Results are presented as mean ± standard error of the mean (SEM). In order to compare the 6 experimental groups, we employed one-factor analysis of variance (ANOVA) followed by the Tukey or the Newman-Keuls post hoc tests. When variances were not homogeneous the nonparametric Kruskal Wallis test was used followed by Mann Whitney post hoc test comparisons. Statistical significance was established with a P value ≤0.05.

Results

Blood Pressure and Antihypertensive Effects

As predicted, 3 weeks after DOCA and salt administration a significant increase in SBP by 33,2 mm Hg compared with the sham group was observed (F = 19,43, P < 0.001, Figure 1A). In the untreated DOCA-salt group SPB continued to rise significantly at week 6 by 70 mm Hg compared to the sham group (Figure 1A).

Figure 1.

Systolic blood pressure and effect of hydrochlorothiazide (HCTZ), fasudil, spironolactone, and fasudil + HCTZ. A, Weekly systolic blood pressure (SBP, mm Hg, mean ± SEM) by the tail cuff method is depicted for the 5 experimental groups. Hydrochlorothiazide, fasudil, and spironolactone were administered for 3 weeks starting on week 3 (arrow) (F = 19.43; P < 0.0001; n = 10/group). Comparisons (post hoc) are shown at weeks 3, 4, 5 and 6. Symbols: **** = P < 0.0001 vs the sham group; ^### = P < 0.001 fasudil vs the untreated DOCA-salt group at week 4; ^#### = P < 0.0001 vs the untreated DOCA-salt group at weeks 5 and 6; & = P < 0.05 hydrochlorothiazide and spironolactone groups vs the untreated DOCA-salt group at week 6. B, Absolute SBP changes within each experimental group from week 3 to week 6 (ΔSBP, mm Hg, mean ± SEM) are depicted and compared with the respective SBP changes observed both in the sham and the untreated DOCA-salt groups (F = 39.17; P < 0.0001; n = 10/group). Symbols: **** = P < 0.0001 vs the sham group; ^#### = P < 0.0001 vs the untreated DOCA-salt group.

Three weeks after drugs administration, SBP was significantly reduced in the 4 treated groups (F = 112,7, P < 0.001, Figure 1A). By comparing with the untreated DOCA salt group, HCTZ administered during 3 weeks reduced significantly SBP by 51.3 mm Hg, similar to the effect due to spironolactone. At the same time (3 week after treatment), compared to the untreated DOCA salt group the antihypertensive effect was larger with the ROCK inhibitor FAS than with HCTZ (by 15.7 mm Hg, P < 0.05) and with spironolactone (by 14.7 mm Hg, P < 0.05) and similar to the antihypertensive effect of combined HCTZ + FAS.

The rate of SBP reduction during the first 2 weeks of treatment was similar in the groups treated with fasudil, HCTZ and with fasudil + HCTZ. Thus, after 1 and 2 weeks of treatment, SBP was similar in the aforementioned 3 groups (Figure 1A).

Within the 4 treatment groups the absolute SBP reduction (difference between weeks 3 and 6) was similar (Figure 1B).

Myocardial Hypertrophy and Fibrosis

Compared to the sham group a significant increase (P < 0.05) was observed in the untreated DOCA-salt group, both in LV area (by 29%) and in LV anterior wall thickness (LVAWT, by 18%) with a significant reduction in LV area in the fasudil, HCTZ and with HCTZ + fasudil treated groups and in LVAWT in the fasudil and HCTZ + fasudil treated groups (Figure 2).

Figure 2.

LV wall sectional area and thickness. A, Representative LV cross sections stained with hematoxylin and eosin in the 6 experimental groups under light microscopy. B-D, LV area (mm²), posterior wall thickness (LVPWT, µm) and anterior wall thickness (LVAWT, µm), respectively. Compared to the sham group a significant increase (P < 0.05) was observed in the untreated DOCA-salt group, both in LV area (by 29%) and in LVAWT (by 18%) with a significant reduction in LV area with fasudil, HCTZ and with HCTZ + fasudil and with fasudil and HCTZ + fasudil in the case of LVAWT; (n = 6 per group; data shown as mean and SEM. Symbols: * = P < 0.05 vs sham group, ^# P < 0.05 vs the untreated DOCA group, comparisons after significant Kruskal Wallis test). E-G, Indexed by body weight (BW) LV area/BW (mm²/gram), posterior wall thickness (LVPWT/BW, µm/gram) and anterior wall thickness (LVAWT/BW, µm/gram), respectively. Compared to the sham group a significant increase (P < 0.05) was observed in the untreated DOCA-salt group, in LV area/BW (by 2.4 fold), LVPWT/BW (by 2.7 fold) and in LVAWT/BW (by 2.1 fold). By contrast, a significant reduction was observed in LV area/BW, LVPWT/BW and in LVAWT/BW in the 4 treated experimental groups (n = 6 per group; data shown as mean and SEM. Symbols: * = P < 0.05 vs sham group, ^# P < 0.05 vs the untreated DOCA group, comparisons after significant Kruskal Wallis test).

When indexed by body weight (BW), a significant increase (P < 0.05) was observed in the untreated DOCA-salt group compared to the sham group in LV area/BW (by 2.4 fold), LV posterior wall thickness (LVPWT/BW, by 2.7 fold) and in LVAWT/BW (by 2.1 fold). By contrast, a significant reduction was observed in LV area/BW, LVPWT/BW and in LVAWT/BW in the 4 treated experimental groups (Figure 2).

In comparison with the sham group, hypertension in the untreated DOCA-Salt group induced myocardial hypertrophy with a significant increase both in cardiomyocyte perimeter and cross-sectional area by 47 and 240%, respectively rats (Figure 3). Cardiomyocyte perimeter was 91.6 ± 1.7 and 62.2 ± 0.3 µm in the untreated DOCA-Salt group and in the sham group, whereas cardiomyocyte area was 549.7 ± 19.1 and 228.8 ± 2.7 µm² in both respective groups (Figure 3). Compared to the untreated DOCA-Salt group, HCTZ treatment reduced significantly cardiomyocyte perimeter and area by 20 and 62% respectively (Figure 3), which was similar to the effect of fasudil and to the combination of fasudil + HCTZ, but less than the effect with spironolactone. Additionally, relative cardiac weight was increased by 73% in the untreated DOCA-Salt group and it was decreased significantly by HCTZ, fasudil, spironolactone and to the combination of fasudil + HCTZ (Figure 3).

Figure 3.

Cardiomyocyte hypertrophy and relative cardiac weight. A, Representative LV cross sections stained with hematoxylin and eosin in the 6 experimental groups under light microscopy at 200×. Compared to the untreated DOCA-salt group, a significant reduction of both cardiomyocyte perimeter (B; F = 138.4; P < 0.0001; n = 10-12 per group) and area (C; F = 139.3; P < 0.0001; n = 10-12 per group) were similarly observed with hydrochlorothiazide, fasudil, spironolactone and with the combination of HCTZ + fasudil. Relative cardiac weight (RCW, g/cm), calculated as the ratio of cardiac weight (g) to the length of the tibia (cm) was significantly reduced with hydrochlorothiazide, fasudil and with spironolactone and with the combination of HCTZ + fasudil (D; F = 23.5; P < 0.0001; n = 9-12 per group); data shown as mean and SEM. Symbols (comparisons after significant ANOVA): ^### = P < 0.001 vs untreated DOCA-salt group; *** = P < 0.001 vs the sham group.

Additionally, myocardial collagen deposits were increased significantly in the untreated DOCA-Salt group (by 6 fold in the whole myocardium, 6.3 fold in the mid myocardium and by 5.4 fold in the sub endocardium, Figure 4). HCTZ, fasudil, spironolactone and the combination of fasudil + HCTZ administered for 3 weeks to DOCA hypertensive rats significantly reduced myocardial collagen deposits to similar levels as in the sham group (Figure 4).

Figure 4.

Myocardial fibrosis. A, Representative LV cross sections stained with picrosirius red in the 6 experimental groups (n = 8-12 per group) under light microscopy at 200× (top) and under polarization light (bottom). Thick collagen fibers take on a yellow or yellow-red color and thin fibers are green when viewed with crossed polarizing filters at light microscopy. B, Collagen volume fraction in the 6 experimental groups is depicted in the sub endocardium (top figure left), (F = 632; P < 0.0001), in the mid myocardium (top figure right) (F = 556; P < 0.0001), and in the whole myocardium (bottom figure) (F = 632; P < 0.0001). Compared to the untreated DOCA-salt group, a significant reduction of myocardial collagen was similarly observed with HCTZ, fasudil, spironolactone and with the combination of HCTZ + fasudil (mean and SEM). Symbols (comparisons after significant ANOVA): ^### = P < 0.001 vs the untreated DOCA-salt group; *** = P < 0.001 vs the sham group. C, Chronically deposited collagen (red yellow fibers) determined as the % of the cross sectional area under polarized light (left) as well as freshly deposited collagen (green fibers) at right. Symbols (comparisons after significant Kruskal-Wallis test): ** = P < 0.001 vs the sham group, ^## = P < 0. 01 and ^# = P < 0.05 vs the untreated DOCA-salt group, respectively.

Under polarized light, chronically deposited collagen (visualized as red—yellow refringence) was significantly increased by 5 fold in the untreated DOCA-Salt group in comparison with the sham group (Figure 4), while HCTZ, fasudil, spironolactone and fasudil + HCTZ administered for 3 weeks to DOCA hypertensive rats significantly reduced chronically myocardial collagen deposits to similar levels as were observed in the sham group (red refringence, Figure 4). By contrast, freshly deposited myocardial collagen levels (visualized as green refringence) were similar in the 6 experimental groups (Figure 4).

Pro-Fibrotic Proteins in the Myocardium

Consistent with the histological and morphometric findings, in the untreated DOCA-Salt group, protein levels of the pro-fibrotic proteins Col-I, Col-III and TGF-β1 were significantly increased compared with the sham rats by 2.3 fold, 3.1 fold and 60%, respectively (P < 0.05; Figure 5). Administration of HCTZ, fasudil, spironolactone and fasudil + HCTZ to DOCA hypertensive rats for 3 weeks prevented the increase in the levels of these 3 pro-fibrotic proteins in the myocardium, which was similar to the levels observed in the sham group (P < 0.05; Figure 5).

Figure 5.

Pro-fibrotic protein levels (Col-I, Col-III and TGF-β1) in the myocardium (Western blot). A, Representative Western blots of Col-I, Col-III and TGF-β1 in the 6 experimental groups. B, Comparative protein levels of Col-I, Col-III and TGF-β1 in the 5 experimental groups. Compared to the untreated DOCA-salt group, a significant reduction of myocardial protein levels of Col-I (F = 3.65; P < 0.01; n = 7-10 per group), Col-III (F = 5.83; P < 0.001; n = 8-10 per group) and TGF-β1 (F = 5.22; P < 0.005; n = 6-8 per group) was observed with hydrochlorothiazide, fasudil, spironolactone as well as with the combination of HCTZ + fasudil (mean and SEM). Symbols (comparisons after ANOVA): ^# = P < 0.05 vs untreated DOCA salt group; ^## = P < 0.01 vs untreated DOCA salt group; ^### = P < 0.001 vs untreated DOCA salt group; *= P < 0.05 vs the sham group; ** = P < 0.01 vs the sham group; *** = P < 0.001 vs the sham group.

Cardiac ROCK Activation

ROCK activity in the myocardium was determined by MYPT1 phosphorylation, a direct ROCK target^26,33,43 as the ratio of phosphorylated to total MYPT1 (p-MYPT1/MYPT1) by Western blot. In the untreated DOCA-Salt group, myocardial ROCK activity was significantly increased by 54% compared to the sham group (Figure 5). HCTZ, fasudil, spironolactone and fasudil + HCTZ administered for 3 weeks reduced myocardial ROCK activation to levels observed in the sham control group (Figure 6).

Figure 6.

Cardiac ROCK activity (determined by MYPT1 phosphorylation, as the ratio of phosphorylated to total MYPT1 (p-MYPT1/MYPT1) by Western blot. Representative Western blots of p-MYPT1 and total MYPT1 in the 6 experimental groups (A). Comparative cardiac ROCK activation in the 6 experimental groups (B). ROCK activation in the untreated DOCA-salt group, was significantly reduced to levels observed in the sham group by hydrochlorothiazide, fasudil, spironolactone and with the combination of HCTZ + fasudil (mean and SEM, F = 4.95; P < 0.005; n = 6-10 per group). Symbols (comparisons after ANOVA): ^# = P < 0.05 vs the untreated DOCA salt group; * = P < 0.05 vs the sham group.

Gene Expression of Pro Remodeling and Pro Oxidative Molecules in the Myocardium

In the untreated hypertensive DOCA salt group, expression of the pro fibrotic genes TGF-β1 and CTGF in the LV myocardium was significantly increased by 75% and 67%, respectively which was significantly prevented/reduced to levels as in the sham group by HCTZ, spironolactone, the ROCK inhibitor fasudil and by the combination of HCTZ + fasudil (Figure 7).

Figure 7.

A, Representative RT-PCR amplification products of cardiac gene expression of TGF-β1, CTGF, MCP-1, PAI-1, COX-2, gp91 phox and p22 phox in the 6 experimental groups. Amplification products are fractionated on a 1.5% agarose gel and visualized by staining with ethidium bromide. B, Comparative myocardial gene expression of TGF-β1 (F = 19.82, P < 0.001), CTGF (F = 17.78, P < 0.001), MCP-1 (F = 7.52, P < 0.001), PAI-1 (F = 23.87, P < 0.0001), COX-2 (F = 8.94, P < 0.001), gp91 (F = 13.24, P < 0.0001) phox and p22 phox (F = 12.65, P < 0.0001) in the 6 experimental groups (mean and SEM). Symbols (comparisons after ANOVA): ^## = P < 0.01 vs untreated DOCA salt group; ^### = P < 0.001 vs untreated DOCA salt group; *= P < 0.05 vs the sham group; ** = P < 0.01 vs the sham group; *** = P < 0.001 vs the sham group. ^$$ = P < 0.01 vs the DOCA-HCTZ group.

Besides, in the untreated DOCA salt hypertensive group myocardial expression of the proinflammatory gene MCP-1 and the procoagulant PAI-1 gene was significantly increased by 21fold and by 40%, respectively. HCTZ, spironolactone, fasudil and HCTZ + fasudil administered for 3 weeks prevented/reduced the increase in the levels of both MCP-1 and PAI-1 cardiac gene expression (Figure 7).

Additionally, myocardial expression of the pro oxidative genes COX2, gp91phox and p22phox was significantly increased in hypertensive DOCA-salt rats compared with the sham group by 9.9 fold, 57% and 2.6 fold, respectively (P < 0.05; Figure 7). HCTZ, spironolactone, fasudil and HCTZ + fasudil administered for 3 weeks to DOCA hypertensive rats significantly prevented/reduced these effects (Figure 7) to similar extent with regard to gp91phox and p22phox genes.

Discussion

The main finding here was that HCTZ, a commonly used thiazide antihypertensive drug, prevented/regressed pathologic LVH in a preclinical model of hypertension by reducing blood pressure, hypertrophy and myocardial fibrosis. In addition, hydrochlorothiazide reduced myocardial Rho kinase activation and levels of the pro-fibrotic proteins Col-I, Col-III and TGF-β1 as well as myocardial gene expression of the pro fibrotic genes TGF-β1 and CTGF, the pro inflammatory gene MCP-1, the procoagulant PAI-1 gene and the pro oxidative genes COX2, gp91phox and p22phox. These observed effects of hydrochlorothiazide in the hypertensive myocardium were similar to those observed with the direct ROCK inhibitor fasudil and with the mineralocorticoid receptor antagonist blocker spironolactone.

There are just a few observations on the effects of hydrochlorothiazide alone on myocardial remodeling in preclinical hypertension, none of them assessing at the same time its effect on myocardial Rho kinase activity. In spontaneously hypertensive rats (SHR), long term oral administration of the angiotensin receptor blocker (ARB) losartan as well as of losartan plus hydrochlorothiazide showed that in the losartan group blood pressure and systolic LV function were maintained, LV mass/BW increased with age and diastolic function was reduced (as in the untreated SHR control group).⁴⁴ Interestingly, in the losartan + HCTZ group, blood pressure was reduced and LV mass/BW as well as systolic and diastolic function were maintained in this group. The progression of myocardial fibrosis was clearly reduced in rats in both groups treated with ARB, concluding that addition of HCTZ proved to be more effective than monotherapy to prevent LVH and diastolic dysfunction.⁴⁴ In the Dahl hypertensive rat losartan, HCTZ and the combination of both drugs administered during 5 weeks systolic blood pressure was reduced with losartan and to a greater extent with HCTZ.⁴⁵ Both drugs reduced LVH, myocardial fibrosis, diastolic dysfunction, as well as gene expression of the angiotensin-converting enzyme and the angiotensin II type 1A receptor similarly. Losartan downregulated expression of the AT1A receptor gene in the aorta. However, HCTZ upregulated the gene expression of the AT2 receptor significantly in the aorta. Additionally, in these experiments plasma concentration of nitric oxide metabolites was reduced by losartan and it was abolished by HCTZ.⁴⁵ In stroke prone SHR rats treated with oral indapamide or with hydrochlorothiazide during 44 days, systolic blood pressure was reduced. Both thiazides, indapamide and hydrochlorothiazide, prevented the development of cardiac hypertrophy, and myocardial fibrosis was virtually absent in the 2 groups.⁴⁶ These observations are in keeping with our current results on the protective effect of HCTZ on LVH as well as on myocardial fibrosis (mainly on chronically deposited collagen as was here visualized under polarized light with picrosirius red staining) in hypertension.

The picrosirius red technique, together with polarization microscopy allows to monitor the transformation of the fibrillar collagen matrix and to examine the manner in which these fibers become entwined during collagen accumulation in the hypertrophied myocardium.⁴⁷ With this dye, thick collagen fibers take on a yellow or yellow-red color while thin fibers are green when viewed with crossed polarizing filters at light microscopy.^48
-50 By using antibody labeling to type I and III collagens, it has been suggested that collagen struts between myocytes in the rat myocardium are type I collagen⁵¹ and endomysial fibers are composed of both type I and III collagens.⁵²

In the aortic wall we previously found that both HCTZ and spironolactone reduced ROCK activation, over expression of profibrotic and pro-oxidative genes and also aortic wall hypertrophy in DOCA salt hypertensive rats in a similar extent as the direct ROCK inhibitor fasudil.²⁶ In the same hypertensive model, the ROCK inhibitor SAR407899 reduced blood pressure, showed a significant improvement of heart function and attenuation of multifocal fibrosis, perivascular fibrosis and leukocyte infiltration in the heart without measurements of ROCK activity.⁵³ The fact that the effects of HCTZ on myocardial ROCK activity and remodeling were here almost identical to those of the direct ROCK inhibitor fasudil, is consistent with the hypothesis that HCTZ reduces cardiac hypertrophy and fibrosis via Rho-Kinase in DOCA-salt hypertension.

Interestingly, in DOCA salt hypertensive rats, ROCK activity determined in circulating leukocytes reflects the activation of this pathway in the myocardium and is related to myocardial remodeling (hypertrophy, fibrosis and inflammation)³³ which contributes to explain the biological significance of increased ROCK activation in circulating leukocytes in clinical hypertension.^54
-56

In the last meta-analysis of randomized trials comparing the effects of the 5 major antihypertensive drug classes on echocardiographic left ventricular (LVM) mass regression, in pairwise comparisons of the 5 drug classes administered during at least 2 months (84 pairwise comparisons, 6001 hypertensive patients), the only significant difference among the drug classes was a lesser regression of LVM due to betablockers (mostly atenolol) than by angiotensin receptor blockers whereas SBP reduction was similar.⁵⁷ Regression of LVM was based on a reduction of wall thickness by all antihypertensive classes and a further reduction of the internal LV diameter by diuretics (hydrochlorothiazide, indapamide, and chlorthalidone).⁵⁷ In a recent systematic review among 28 double blind clinical trials in hypertension, HCTZ reduced significantly LVM by −7.3%⁵⁸ and other diuretics significantly surpassed HCTZ in LVM reduction: chlorthalidone by −8.2% and indapamide by −7.5%, and potassium-sparing/hydrochlorothiazide combinations by −7.7% which was not significant.⁵⁸ In this systematic review the blood lowering effect was similar among all diuretics.

In 371 hypertensive patients with diastolic dysfunction and not achieving their therapeutic goals by using an ACEi or an ARB, the change from these single antihypertensive drugs to the combination of losartan + HCTZ during 6 months was related to BP reduction, LV relaxation improvement, reduction of systemic inflammation as well as to improvement in the heart failure state.⁵⁹ In this study the effect of adding HCTZ on myocardial remodeling, as observed in our current experimental study, possibly favored improvement in LV relaxation.

Spironolactone, the standard non-selective mineralocorticoid receptor antagonist was used here as a well proved antihypertensive and anti-myocardial remodeling comparator agent.⁴⁰ In humans, spironolactone is used in resistant hypertension which is generally volume-dependent due to varying degrees of aldosterone excess.⁴¹ In contrast to spironolactone, which possesses anti-androgenic side effects, eplerenone is a more recently discovered aldosterone inhibitor, with higher specificity for aldosterone receptors and not evident endocrine action. However, because of the current evidence provided by the results of the PATHWAY-2⁶⁰ and ReHOT⁶¹ clinical studies, spironolactone has been established as the most effective add-on anti-aldosterone therapy in resistant hypertension.⁴¹

The effect of aldosterone on ROCK activation has been assessed in clinical and preclinical studies. In hypertensive patients, the mineralocorticoid (MR) receptor blocker eplerenone reduces blood pressure, improves endothelial function, and inhibits ROCK activity⁶² and in patients with hyperaldosteronism it improves endothelial function, arterial stiffness and inhibits ROCK activity.⁶³ In obesity-associated nephropathy in C57BL/6 J mice, renal injury is mediated by mineralocorticoid receptor activation in the kidney and subsequent ROCK stimulation.⁶⁴ Furthermore, in obese db/db mice with hyperaldosteronism there is vascular MR hyperactivation, dysfunction and remodeling, increased ROCK signaling and higher expression of pro-inflammatory and pro-fibrotic markers.⁶⁵ These effects are reduced by the MR antagonist canreonate and with fasudil indicating an interplay between the MR and ROCK inducing pro-contractile, pro-inflammatory and pro-fibrotic processes that trigger diabetes-related vascular changes.⁶⁵ The aforementioned processes could be simultaneously occurring in the myocardium as is observed in other preclinical models of cardiovascular remodeling and ROCK activation^33,43 and could explain here the similar observed protective effects of spironolactone, fasudil and also of HCTZ—by reducing ROCK activation—on myocardial hypertensive remodeling.

The current findings, specifically LVH reduction also explain the ability of thiazides to prevent heart failure in hypertensive patients. In this regard, in a recent analysis of the ALLHAT clinical study with high-risk hypertensive patients comparing the thiazide chlorthalidone with doxazosin, amlodipine, and also with lisinopril, it was found that evolving LVH and blood pressure lowering explain up to 13% of the HF-preventive effect of chlorthalidone as compared with the preventive effect of antihypertensive treatment with the 3 other drugs.⁶⁶ In addition to BP lowering, thiazides such as chlorthalidone have pleiotropic effects including improving endothelial function and reducing inflammation and oxidative stress,⁶⁷ and some of these pleiotropic effects might be mediated by reduction of Rho kinase activity as was found here.

Limitations

One question not completely addressed here was whether the effect of hydrochlorothiazide on ROCK activation and myocardial remodeling was due only to blood pressure reduction and secondary afterload reduction. In this regard, the effects of this thiazide, of the ROCK inhibitor fasudil as well as the effect of both agents combined were similar, as were the effects of spironolactone. In all available studies in experimental hypertension assessing myocardial remodeling and Rho kinase activation, blood pressure has also been reduced. Based on our current findings, we cannot rule out that protective effects of HTCZ and spironolactone on ROCK activity are not specific to these agents and are attributable to blood pressure fall by them. In this sense, future studies are necessary to dilucidate whether the effect of HCTZ on cardiac remodeling via ROCK depends or not on blood pressure reduction, but current evidence suggests that both effects are participating. Thus, whether the effect of hydrochlorothiazide on ROCK activity are direct or not and whether the protective effect requires chronic treatment or not should be selectively characterized in future experiments.

In addition, some relevant mechanisms involved in hypertensive hypertrophy and consequent cardiac remodeling in which the ROCK pathway is significantly implicated were not here assessed such as cardiac hypertrophy-related Ca2+ dysregulation,³⁷ cardiomyocyte apoptosis, cardiac fetal gene expression, activation of myocardin-related transcription factor (MRTF) and serum response factor (SRF) which induces expression of profibrotic gene in myofibroblasts as well as fibroblasts to myofibroblasts differentiation.³⁹ Besides, we have not analyzed coronary microvascular dysfunction and ischemia probably due to oxidative stress, enhanced vasoconstriction to endothelin 1 and reduced endothelial NO function through increased ROCK signaling.³⁸

In summary, hydrochlorothiazide reduced pathologic LVH in a preclinical model of hypertension by controlling blood pressure, hypertrophy and myocardial fibrosis as well as by reducing myocardial ROCK activation, which was associated to reduced myocardial expression of pro remodeling, pro fibrotic and pro oxidative genes. These observed effects of hydrochlorothiazide in the hypertensive myocardium were similar to those observed with the direct ROCK inhibitor fasudil (and also with the aldosterone receptor blocker spironolactone) and can explain some preventive effects of thiazides in clinical hypertension, specifically LVH regression and incident heart failure.

Footnotes

Author Contributions

David Mondaca-Ruff and Patricio Araos contributed equally to the article.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the Agencia Nacional de Investigacion y Desarrollo (ANID, Chile): Fondecyt (Fondo Nacional de Desarrollo Cientifico y Tecnologico, Chile) 1161739 (to J.E.J) and 3201016 (to P.A), Anillo ACT192144 (to M.P.O and J.E.J) and FONDAP 15130011 (to M.P.O).

ORCID iDs

Patricio Araos

Jorge E. Jalil

References

Whelton

Carey

Aronow

, et al. 2017 ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA guideline for the prevention, detection, evaluation, and management of high blood pressure in adults: a report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. Circulation. 2018;71(6):e484–594.

Mills

Bundy

Kelly

, et al. Global disparities of hypertension prevalence and control: a systematic analysis of population-based studies from 90 countries. Circulation. 2016;134(6):441–450.

GBD 2017 Risk Factor Collaborators. Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990-2017: a systematic analysis for the global burden of disease study 2017. Lancet. 2018;392(10159):1923–1994.

GBD 2017 Causes of Death Collaborators. Global, regional, and national age-sex-specific mortality for 282 causes of death in 195 countries and territories, 1980-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. 2018;392(10159):1736–1788.

Cohn

Ferrari

Sharpe

. Cardiac remodeling-concepts and clinical implications: a consensus paper from an international forum on cardiac remodeling. Behalf of an International Forum on Cardiac Remodeling. J Am Coll Cardiol. 2000;35(3):569–582.

Celic

Tadic

Suzic-Lazic

, et al. Two- and three-dimensional speckle tracking analysis of the relation between myocardial deformation and functional capacity in patients with systemic hypertension. Am J Cardiol. 2014;113(5):832-839.

Tadic

Majstorovic

Pencic

, et al. The impact of high-normal blood pressure on left ventricular mechanics: a three-dimensional and speckle tracking echocardiography study. Int J Cardiovasc Imaging. 2014;3(4):699-711

Devereux

Wachtell

Gerdts

, et al. Prognostic significance of left ventricular mass change during treatment of hypertension. JAMA. 2004;292:2350-2356.

Muiesan

Salvetti

Paini

, et al. Inappropriate left ventricular mass changes during treatment adversely affects cardiovascular prognosis in hypertensive patients. Hypertension. 2007;49(5):1077-1083.

10.

Okin

Hille

Kjeldsen

Dahlöf

Devereux

. Persistence of left ventricular hypertrophy is associated with increased cardiovascular morbidity and mortality in hypertensive patients with lower achieved systolic pressure during antihypertensive treatment. Blood Press. 2014;23(2):71-80.

11.

Antikainen

Peters

Beckett

, et al. Left ventricular hypertrophy is a predictor of cardiovascular events in elderly hypertensive patients: hypertension in the very elderly trial. J Hypertens. 2016;34(11):2280-2286.

12.

Weir

Townsend

. What is left ventricular hypertrophy and is there a reason to regress left ventricular hypertrophy. J Clin Hypertens. 2009;11(8):407-410.

13.

Pierdomenico

Cuccurullo

. Risk reduction after regression of echocardiographic left ventricular hypertrophy in hypertension: a meta-analysis. Am J Hypertens. 2010;23(8):876-881.

14.

Williams

Mancia

Spiering

, et al. 2018 practice guidelines for the management of arterial hypertension of the European Society of Hypertension and the European Society of Cardiology: ESH/ESC Task Force for the Management of Arterial Hypertension. J Hypertens. 2018;36(10):2284–2309.

15.

James

Oparil

Carter

, et al. 2014 evidence-based guideline for the management of high blood pressure in adults: report from the panel members appointed to the Eighth Joint National Committee (JNC 8). JAMA. 2014;311(5):507–520.

16.

ALLHAT Officers and Coordinators for the ALLHAT Collaborative Research Group. The antihypertensive and lipid-lowering treatment to prevent heart attack trial. Major outcomes in high-risk hypertensive patients randomized to angiotensin-converting enzyme inhibitor or calcium channel blocker vs diuretic: the antihypertensive and lipid-lowering treatment to prevent heart attack trial (ALLHAT). JAMA. 2002;288(23):2981–2997.

17.

Suchard

Schuemie

Krumholz

, et al. Comprehensive comparative effectiveness and safety of first-line antihypertensive drug classes: a systematic, multinational, large-scale analysis. Lancet. 2019;394(10211):1816–1826.

18.

Herman

Bashir

. Hydrochlorothiazide. In: StatPearls [Internet]. StatPearls Publishing; 2021. PMID: 28613517.

19.

Tamargo

Segura

Ruilope

. Diuretics in the treatment of hypertension. Part 1: thiazide and thiazide-like diuretics. Expert Opin Pharmacother. 2014;15(4):527–547.

20.

Pickkers

Hughes

Russel

, et al. Thiazide-induced vasodilation in humans is mediated by potassium channel activation. Hypertension. 1998;32:1071–1076.

21.

Calder

Schachter

Sever

. Potassium channel opening properties of thiazide diuretics in isolated guinea pig resistance arteries. J Cardiovasc Pharmacol. 1994;24:158–164.

22.

Pickkers

Garcha

Schachter

, et al. Inhibition of carbonic anhydrase accounts for the direct vascular effects of hydrochlorothiazide. Hypertension. 1999;33(4):1043–1048.

23.

Colas

Slama

Collin

, et al. Mechanisms of methyclothiazide-induced inhibition of contractile responses in rat aorta. Eur J Pharmacol. 2000;408(1):63–67.

24.

Calder

Schachter

Sever

. Potassium channel opening properties of thiazide diuretics in isolated guinea pig resistance arteries. J Cardiovasc Pharmacol. 1994;24(1):158–164.

25.

Zhu

Liu

, et al. Thiazide-like diuretics attenuate agonist induced vasoconstriction by calcium desensitization linked to Rho kinase. Hypertension. 2005;45(2):233–239.

26.

Araos

Mondaca

Jalil

, et al. Diuretics prevent Rho-kinase activation and expression of profibrotic/oxidative genes in the hypertensive aortic wall. Ther Adv Cardiovasc Dis. 2016;10(6):338–347.

27.

Strassheim

Gerasimovskaya

Irwin

Dempsey

Stenmark

Karoor

. RhoGTPase in vascular disease. Cells. 2019;8(6):551. doi:10.3390/cells8060551 PMID: 31174369; PMCID: PMC6627336

28.

Shimokawa

Sunamura

Satoh

. RhoA/Rho-kinase in the cardiovascular system. Circ Res. 2016;118(2):352–366.

29.

Masumoto

Hirooka

Shimokawa

Hironaga

Setoguchi

Takeshita

. Possible involvement of Rho-kinase in the pathogenesis of hypertension in humans. Hypertension. 2001;38(6):1307–1310.

30.

Soga

Noma

Hata

, et al. Rho-associated kinase activity, endothelial function, and cardiovascular risk factors. Arterioscler Thromb Vasc Biol. 2011;31(10):2353–2359.

31.

Smith

Santhanam

Alexander

. Rho-kinase activity and cutaneous vasoconstriction is upregulated in essential hypertensive humans. Microvasc Res. 2013;87:58–64.

32.

Gabrielli

Winter

Godoy

, et al. Increased Rho-kinase activity in hypertensive patients with left ventricular hypertrophy. Am J Hypertens. 2014;27(6):838–845.

33.

Ocaranza

Fierro

Jalil

, et al. Rho kinase activation in circulating leukocytes is related to hypertensive myocardial remodeling. Clin Sci (Lond). 2018;132(16):1837–1853.

34.

Uehata

Ishizaki

Satoh

, et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature. 1997;389(6654):990–994.

35.

Ocaranza

Rivera

Novoa

, et al. Rho kinase inhibition activates the homologous angiotensin-converting enzyme-angiotensin-(1-9) axis in experimental hypertension. J Hypertens. 2011;29(4):706–715.

36.

Dhaliwal

Casey

Greco

, et al. Rho kinase and Ca2⁺ entry mediate increased pulmonary and systemic vascular resistance in L-NAME-treated rats. Am J Physiol Lung Cell Mol Physiol. 2007;293(5):L1306–L1313.

37.

Olgar

Celen

Yamasan

Ozturk

Turan

Ozdemir

. Rho-kinase inhibition reverses impaired Ca²⁺ handling and associated left ventricular dysfunction in pressure overload-induced cardiac hypertrophy. Cell Calcium. 2017;67:81–90.

38.

Tsai

Ren

Hein

Kuo

. Enhanced endothelin-1/Rho-kinase signalling and coronary microvascular dysfunction in hypertensive myocardial hypertrophy. Cardiovasc Res. 2017;113(11):1329–1337.

39.

Shimizu

Liao

. Rho kinases and cardiac remodeling. Circ J. 2016;80(7):1491–1498.

40.

Ferreira

Rossignol

Pizard

, et al. Potential spironolactone effects on collagen metabolism biomarkers in patients with uncontrolled blood pressure. Heart. 2019;105(4):307–314.

41.

Manolis

Melita

Manolis

. Eplerenone versus spironolactone in resistant hypertension: an efficacy and/or cost or just a men’s issue? Curr Hypertens Rep. 2019;21(3):22. doi:10.1007/s11906-019-0924-0

42.

Morais

Viana

Silva

FMO

, et al. Polarization microscopy as a tool for quantitative evaluation of collagen using picrosirius red in different stages of CKD in cats. Microsc Res Tech. 2017;80(5):543–550.

43.

Fierro

Novoa

González

Ocaranza

Jalil

. Simultaneous Rho kinase inhibition in circulating leukocytes and in cardiovascular tissue in rats with high angiotensin converting enzyme levels. Int J Cardiol. 2016;215:309–317.

44.

Masuda

Taenaka

Asanuma

Nakatani

. Comparison of the effects of angiotensin II receptor antagonist monotherapy and combination therapy with a diuretic on cardiac function in spontaneously hypertensive rats. J Echocardiogr. 2012 Dec;10(4):125–131.

45.

Yamada

Tsuboi

Hattori

, et al. Mechanism underlying the efficacy of combination therapy with losartan and hydrochlorothiazide in rats with salt-sensitive hypertension. Hypertens Res. 2011;34(7):809–816.

46.

Contard

Glukhova

Marotte

, et al. Diuretic effects on cardiac hypertrophy in the stroke prone spontaneously hypertensive rat. Cardiovasc Res. 1993;27(3):429–434.

47.

Jalil

Doering

Janicki

Pick

Shroff

Weber

. Fibrillar collagen and myocardial stiffness in the intact hypertrophied rat left ventricle. Circ Res. 1989;64(6):1041–1050.

48.

Junqueira

LCU

Cossermelli

Brentani

. Differential staining of collagens type I, II, and III by Sirius Red and polarization microscopy. Arch Histol Jpn. 1978;41(3):267–274.

49.

Jalil

Doering

Janicki

, et al. Structural vs. contractile protein remodeling and myocardial stiffness in hypertrophied rat left ventricle. J Mol Cell Cardiol. 1988;20(12):1179–1187.

50.

Pick

Jalil

Janicki

Weber

. The fibrillar nature and structure of isoproterenol-induced myocardial fibrosis in the rat. Am J Pathol. 1989;134(2):365–371.

51.

Robinson

Factor

Capasso

Wittenberg

Blumenfeld

Seifter

. Morphology, composition, and function of struts between cardiac myocytes of rat and hamster. Cell Tissue Res. 1987;249(2):247–255.

52.

Robinson

Cohen-Gould

Factor

Eghbali

Blumenfeld

. Structure and function of connective tissue in cardiac muscle: collagen types I and III in endomysial struts and pericellular fibers. Scanning Microsc. 1988;2(2):1005–1015.

53.

Löhn

Plettenburg

Kannt

, et al. End-organ protection in hypertension by the novel and selective Rho-kinase inhibitor, SAR407899. World J Cardiol. 2015;7(1):31–42.

54.

Hata

Soga

Hidaka

, et al. Calcium channel blocker and Rho-associated kinase activity in patients with hypertension. J Hypertens. 2011;29(2):373–379.

55.

Cantin

Jalil

Bulnes

, et al. Effect of early normotension with olmesartan on rho-kinase activity in hypertensive patients. Curr Vasc Pharmacol. 2020;18(1):87–91.

56.

Ravarotto

Pagnin

Maiolino

, et al. The blocking of angiotensin II type 1 receptor and RhoA/Rho kinase activity in hypertensive patients: effect of olmesartan medoxomil and implication with cardiovascular-renal remodeling. J Renin Angiotensin Aldosterone Syst. 2015;16(4):1245–1250.

57.

Fagard

Celis

Thijs

Wouters

. Regression of left ventricular mass by antihypertensive treatment: a meta-analysis of randomized comparative studies. Hypertension. 2009;54(5):1084–1091.

58.

Roush

Abdelfattah

Song

Kostis

Ernst

Sica

. Hydrochlorothiazide and alternative diuretics versus renin-angiotensin system inhibitors for the regression of left ventricular hypertrophy: a head-to-head meta-analysis. J Hypertens. 2018;36(6):1247–1255.

59.

Ito

Ishii

Kihara

, et al. Effect of ARB/diuretics on diastolic function in patients with hypertension (EDEN) trial investigators. Adding thiazide to a renin-angiotensin blocker improves left ventricular relaxation and improves heart failure in patients with hypertension. Hypertens Res. 2012;35(1):93–99.

60.

Williams

MacDonald

Morant

, et al. Spironolactone versus placebo, bisoprolol, and doxazosin to determine the optimal treatment for drug-resistant hypertension (PATHWAY-2): a randomised, double-blind, crossover trial. Lancet. 2015;386(10008):2059–2068.

61.

Krieger

Drager

Giorgi

DMA

, et al. Spironolactone versus clonidine as a fourth-drug therapy for resistant hypertension: the ReHOT randomized study (resistant hypertension optimal treatment). Hypertension. 2018;71(4):681–690.

62.

Fujimura

Noma

Hata

, et al. Mineralocorticoid receptor blocker eplerenone improves endothelial function and inhibits Rho-associated kinase activity in patients with hypertension. Clin Pharmacol Ther. 2012;91(2):289–297.

63.

Kishimoto

Oki

Maruhashi

, et al. Eplerenone improves endothelial function and arterial stiffness and inhibits Rho-associated kinase activity in patients with idiopathic hyperaldosteronism: a pilot study. J Hypertens. 2019;37(5):1083–1095.

64.

Tokuyama

Wakino

Hara

, et al. Role of mineralocorticoid receptor/Rho/Rho-kinase pathway in obesity-related renal injury. Int J Obes (Lond). 2012;36(8):1062–1071.

65.

Nguyen Dinh Cat

Callera

Friederich-Persson

, et al. Vascular dysfunction in obese diabetic db/db mice involves the interplay between aldosterone/mineralocorticoid receptor and Rho kinase signaling. Sci Rep. 2018;8(1):2952.

66.

Johnson

Oparil

Davis

Tereshchenko

. Prevention of heart failure in hypertension-disentangling the role of evolving left ventricular hypertrophy and blood pressure lowering: the ALLHAT study. J Am Heart Assoc. 2019;8(8):e011961.

67.

Roush

Buddharaju

Ernst

Holford

. Chlorthalidone: mechanisms of action and effect on cardiovascular events. Curr Hypertens Rep. 2013;15(5):514–521.