Abstract
Objective:
Diuretics, including thiazides and thiazide-like drugs, are commonly recommended for treating hypertension, though their precise mechanism of action is not fully understood. This study aimed to investigate the pharmacological effects of trichloromethiazide (TCM) in malignant stroke-prone spontaneously hypertensive rats (M-SHRSP).
Methods:
M-SHRSPs were treated with varying doses of TCM. Prognosis, histological changes, and mRNA expression related to hypertension and stroke were assessed.
Results:
The high-dose TCM group (3%) exhibited significantly lower SBP compared with the untreated group, whereas the low-dose group (0.3%) did not show a significant reduction in SBP. The survival rate was 54% in the low-dose group, whereas all rats in the high-dose group survived without experiencing a stroke by 16 weeks of age. Organ weights in both TCM-treated groups were lower than those in the control group, without severe histological abnormalities, including stroke and sclerosis. Plasma levels of thiobarbituric acid-reactive substances (TBARS) were significantly reduced in both TCM-treated groups. Additionally, 20 genes related to tissue protection, repair, proliferation, maintenance, and function were significantly expressed.
Conclusion:
TCM administration in M-SHRSPs significantly modulated the expression of 20 genes associated with tissue protection and maintenance, and reduced plasma TBARS levels. These findings suggest that TCM, a thiazide diuretic, may protect against tissue impairment in hypertension by modulating gene expression and exhibiting antioxidant activity.
Keywords: antioxidant, diuretics, DNA microarray, expressed messenger RNA, hypertension, hypertension, malignant stroke-prone spontaneously hypertensive rats, thiazide, trichlormethiazide
INTRODUCTION
Hypertension is one of the most prevalent diseases affecting individuals with modern lifestyles. If left untreated, it can lead to life-threatening conditions associated with atherosclerosis, such as myocardial infarction, renal failure, and stroke [1–3]. Global guidelines for hypertension therapy have been established to address these issues. The ACC/AHA 2017 guideline recommends lowering blood pressure to below 130/80 mmHg through quantitative risk assessment [4]. Similarly, the ESH/ESC 2018–2023 and JSH 2019 guidelines recommend maintaining blood pressure at 140/90 mmHg using antihypertensive treatment [5–7]. These treatments typically include thiazide diuretics (TZDs), calcium channel blockers (CCBs), angiotensin-converting enzyme inhibitors (ACEIs), β-blockers, angiotensin II receptor blockers (ARBs), and combinations of these drugs. Despite these established guidelines, the precise mechanisms through which thiazides and thiazide-like drugs exert their antihypertensive effects remain unclear. A systematic review and meta-analysis by Reinhart et al.[8] in 2023, titled ‘First-line diuretics versus other classes of antihypertensive drugs for hypertension’, compared the effects of first-line diuretics with those of other first-line antihypertensive treatments. This review concluded that thiazides and thiazide-like drugs likely do not change total mortality but may decrease some morbidity outcomes, such as cardiovascular events, compared with other drugs. However, the underlying mechanisms are not well understood [8]. To address this gap, we treated malignant stroke-prone spontaneously hypertensive rats (M-SHRSPs) [9] with trichloromethiazide (TCM), a thiazide diuretic.
This study aimed to explore the detailed pharmacological effects of TCM on hypertension and stroke by observing prognosis, histological findings, and mRNA expression in several organs. The changes in mRNA expression because of TCM administration were measured in the mesenteric artery, and renal cortex, which are most affected by blood pressure. Tests were carried out at two stages: at 6 weeks of age for a 2-week short-term administration, and at 9 weeks of age for a 5-week sub-long-term administration.
M-SHRSPs exhibit high blood pressure (> 230 mmHg) at 10 weeks of age and a stroke incidence rate exceeding 95% [10]. As the details of the action characteristics of thiazides have not been clarified, it was planned to clarify the action of TCM given to severe hypertensive model rats (M-SHRSP) by observing prognosis of hypertension and histological findings, and by measuring mRNA expression.
Therefore, a detailed pharmacological analysis using M-SHRSPs is considered an appropriate experimental approach.
MATERIALS AND METHODS
Animals and experimental design
Four-week-old male M-SHRSPs (total number is 84 rats = 14 rats × 3 groups × 2 stages) were purchased from the Kindai University Animal Center (Osaka, Japan) and kept under controlled light conditions (12 h light/dark cycles) at a temperature of 22 ± 2 °C and 60% humidity. The M-SHRSP were divided into three groups at random: one group was fed SP-chow (Funabashi Farm, Chiba, Japan) containing 0.3% TCM, another group was fed SP-chow containing 3% TCM, and a control group was fed SP-chow without any drug. The composition of SP-chow was 28.1% protein, 6% fat, 48.8% carbohydrate, and 0.4% NaCl. The feed was adjusted to 0.8–1 g/10 g body weight/day to eat all food. The experiments were conducted in two stages. In the first stage, observational experiments were conducted with 14 rats in each of the three groups, from 4 to 16 weeks of age. In the second stage, three rats from each group were used for genetic measurements at 6 and 9 weeks of age, and six rats from each group were sacrificed at 12 weeks of age for plasma TBARS measurement and tissue observation. Normotensive Wistar–Kyoto (WKY) rats were used as normal controls for histological examination. Rats were fed their respective diets for 12 weeks with ad libitum access to tap water. TCM (C8H8Cl3N3O4S2; MW: 389.66) was purchased from Shionogi Pharmaceutical Co., Ltd. (Osaka, Japan). All rats were weighed, and systolic arterial blood pressure (SBP) was measured weekly using the tail-cuff method with a photoelectric detector. Animals that died during the study period were immediately necropsied to determine the cause of death, including stroke. Stroke was diagnosed by opening the brain to detect bleeding or an infarct. At 6 and 9 weeks of age, three rats from each group were sacrificed to collect the mesenteric artery, which is one of the main arteries, and the renal cortex, which is the main action point of diuretics and a major organ impaired in hypertension, respectively. At 12 weeks of age, after 8 weeks of treatment, six rats from each group were anesthetized with sodium pentobarbital (50 mg/kg, i.p.; Dainippon Sumitomo Pharmacy, Osaka, Japan), followed by blood sample collection and autopsy for histological examination. Procedures involving animals and their care were conducted according to the guidelines of the Japanese Association for Laboratory Animal Science, with the approval of the Animal Care and Use Committee of Kindai University (Approval No. KAME19-095).
Measurements of antioxidant activities in plasma
Blood samples collected from the descending aorta at 12 weeks of age were heparinized, centrifuged, and stored at −80 °C until analysis. Plasma thiobarbituric acid-reactive substances (TBARS), used as an index of oxidative stress, were measured using a TBARS Assay Kit (Cayman, Michigan, USA) [11].
Histological studies of the kidney, brain, and heart changes
The body and tissue weights of the kidneys, brain, and heart of the rats at 12 weeks of age were compared among the three groups. Tissue weights were expressed as a percentage of body weight (g/100 g body weight) and presented as mean ± SEM (n = 6). The kidney, brain, and heart tissues were excised, cut longitudinally using fine surgical scissors, fixed with 10% formalin neutral buffer solution (Wako Chemicals, Osaka, Japan), embedded in paraffin wax, sliced to 2–3 μm thickness, and mounted on glass slides. Specimens were stained using hematoxylin and eosin (H&E) and Elastica-van Gieson (EVG). Histological changes were observed and photographed using a microscope at 50×, 200×, and 400× magnification.
DNA microarray analysis of the mesenteric artery and renal cortex
Small tissue pieces from the mesenteric artery and renal cortex of three rats per group were homogenized at a pitch speed of 22 strokes/s for 2 min (twice) in a 2 ml plastic tube with 5 mm diameter glass beads using a Qiagen Tissue Lyser (Retsch GmbH & Co., Haan, Germany). Total RNA was extracted using the RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany). RNA quality was evaluated using a RNA Nano Chip and Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbornn, Germany), and RNA of sufficient quality was used for microarray experiments. After quality checks, microarray experiments using the Whole Rat Genome Microarray (4×44k format, Agilent Technologies) were performed. The data were extracted, and the overall raw signal intensities on each array were normalized to median value of all rat probes with the BRB-Array Tools-software version,3.7.0 (Biometric Research Branch) [12]. We used the Benjamini–Hochberg procedure to control the false discovery rate (FDR) less than 0.05. Genes with expression changes greater than two-fold (upregulation) or less than half (downregulation) were selected at the first step. Detailed methods are provided in ref. [13].
Statistical analyses
All results are expressed as mean ± standard error of the mean (SEM). Comparisons among the means of multiple groups were performed using one-way ANOVA followed by the Tukey–Kramer multiple comparison test. Differences were considered statistically significant at P less than 0.05. To compare the survivability between three groups, we performed a log-rank test (Mantel–Cox test). To account for multiple comparisons, a Bonferroni correction was applied to the P values in this analysis.
RESULTS
Blood pressure and body weight
The SBP of M-SHRSP rats at 4 weeks of age did not significantly differ between the groups (Fig. 1). At 10 weeks of age, the SBP was 259.2 ± 8.7 mmHg in the control group, 255.3 ± 4.7 mmHg in the 0.3% TCM group, and 233.6 ± 4.7 mmHg in the 3% TCM group. The 3% TCM group showed significantly lower SBP values than the control group from 6 to 10 weeks of age (P < 0.05), whereas the 0.3% TCM group did not differ significantly from the control group throughout the study period.
FIGURE 1.
SBPs in the groups of control (•), 0.3% trichloromethiazide (▴), and 3% trichloromethiazide (▪) given to malignant stroke-prone spontaneously hypertensive rats measured by the tail-cuff method. Three consecutive SBP readings were obtained in the morning after warming the body to 35 °C for 5 min in a heater box. SBP values (mmHg) in three groups of rats (14 rats each) measured at 4–15 weeks of age were obtained, except for 14–15 weeks in the control group because of high mortality after 12 weeks of age. Values are expressed as the mean ± SEM (n = 6–14). Differences between the control and TCM groups were considered statistically significant at P < 0.05 (∗). M-SHRSP, malignant stroke-prone spontaneously hypertensive rats; TCM, trichloromethiazide.
The body weights of M-SHRSP rats at 6 weeks of age were 155 ± 8.4, 146.5 ± 8.1, and 146.5 ± 8.1 g/rat in control, 0.3% TCM, and 3% TCM groups, respectively. At 10 weeks of age, body weights were 238.5 ± 7.4, 241.5 ± 5.8, and 234.1 ± 7.3 g/rat in control, 0.3% TCM, and 3% TCM groups, respectively. At 12 weeks of age, body weights were 235.8 ± 8.5, 262 ± 14.4, and 263.4 ± 8.1 g/rat in control, 0.3% TCM, and 3% TCM groups, respectively. Thus, there was no difference in body weights between the groups at 4–10 weeks of age, but the control group showed significantly lower weights compared with the 3% TCM group (P < 0.05) at 12 weeks of age.
Stroke incidences from 4 to 16 weeks of ages
Fourteen rats were observed per group. After 10 weeks, stroke-related deaths began in the control group, with all rats dying by 14 weeks (Fig. 2). In the low-dose TCM group, 54% of the rats survived, whereas all rats in the high-dose TCM group survived until 16 weeks. The survivability was significantly different among all three groups (P < 0.001).
FIGURE 2.
Cumulative stroke incidences (%) in control, 0.3% trichloromethiazide, and 3% trichloromethiazide groups of malignant stroke-prone spontaneously hypertensive rats. M-SHRSPs in three groups (control, 0.3% TCM, and 3% TCM) were raised from 4 weeks old to 16 weeks old. Rats that died spontaneously during this period were autopsied to determine the cause of death as stroke. Stroke-related deaths began to occur after 10 weeks of age, and all rats in the control group died by 14 weeks of age. Each group initially included 14 rats. The survivability was significantly different among all three groups (P < 0.001). M-SHRSP, malignant stroke-prone spontaneously hypertensive rats; TCM, trichloromethiazide.
Tissue weights of kidney, brain, and heart per 100 g body weight
Figure 3 shows the kidney, brain, and heart weights per 100 g of body weight. The organ weights in both the 0.3 and 3% TCM groups were lower than those in the control group (P < 0.05). Regarding the heart weight/body weight ratio as shown in Fig. 3(c), one reason why the control group showed a larger increase compared with the 0.3% TCM group regardless of no difference in actual heart weights was because the control group could not gain body weight after 10 weeks of age.
FIGURE 3.
Tissue weights of the kidney (a), brain (b), and heart (c) in control, 0.3% trichloromethiazide, and 3% trichloromethiazide-treated malignant stroke-prone spontaneously hypertensive rats sacrificed at 12 weeks of age. Three groups of M-SHRSPs were raised from 4 to 12 weeks of age in the second stage of the experiment. At 12 weeks, rats from the control group, 0.3% TCM group, and 3% TCM group were sacrificed, and their tissue weights were measured. Tissue weights (g) are expressed as a percentage of 100 g body weight and presented as mean ± SEM (n = 6). Differences between the control and TCM groups were considered statistically significant at P < 0.05 (∗). M-SHRSP, malignant stroke-prone spontaneously hypertensive rats; TCM, trichloromethiazide.
Indices representing oxidative stress in plasma
At 12-weeks, TBARS levels (μmol/l), an index of oxidative stress, were 17.4 ± 0.6 in the control group, 11.4 ± 0.9 in the 0.3% TCM group, and 11.3 ± 0.6 in the 3% TCM group (n = 6). TCM at both doses significantly reduced oxidative stress indices (P < 0.05 versus control group).
Histological changes in kidney, brain and heart tissues with 3% trichloromethiazide intake
In the control group, glomerular sclerosis, thickened basement membranes, and an increase in mesangial cells were observed in the kidneys at 12 weeks. Additionally, lymphocyte and fibroblast infiltration were prominent around the glomeruli, renal tubules, and arteries (Fig. 4). The intercellular spaces were wider in the control group than in the TCM group. In contrast, the kidneys of the TCM group rats showed no abnormal changes. EVG staining, used to analyze collagen and other connective tissues, revealed more collagen fibers in the intercellular spaces of the control group compared with the 3% TCM and WKY groups (Fig. 5). In the brain, the control M-SHRSP group exhibited small-to-moderate bleeding and thrombotic lesions scattered in the subcortical area, with a large number of leukocyte granule cells infiltrating the lesions. However, no such impairments were observed in the brains of the 3% TCM group. EVG staining of heart tissues showed that collagen fibers surrounding the arteries and muscle cells were more prevalent in the control M-SHRSP group compared with the 3% TCM and WKY groups. These were not described quantitively, but in clear histological results.
FIGURE 4.
Histological findings of the kidney stained with hematoxylin and eosin (H&E), comparing normal and 3% trichloromethiazide-treated malignant stroke-prone spontaneously hypertensive rats. Two samples from the control and 3% TCM groups were used to compare histological changes. Images were observed with a microscope at three magnifications: low (50×) magnification (scale bar = 500 μm) on the left, 200× magnification (scale bar = 100 μm) in the middle, and 400× magnification (scale bar = 10 μm) on the right. M-SHRSP, malignant stroke-prone spontaneously hypertensive rats; TCM, trichloromethiazide.
FIGURE 5.
Histological findings of the kidney stained by Elastica-von Gieson (EVG), comparing control and 3% trichloromethiazide-treated malignant stroke-prone spontaneously hypertensive rats, and normotensive Wistar–Kyoto rats. Photographs show the findings of the control M-SHRSP group (upper row), 3% TCM group (middle row), and normotensive WKY rats (lower row). Images were observed with a microscope at three magnifications: low (50×) magnification (scale bar = 500 μm) on the left, 200× magnification (scale bar = 100 μm) in the middle, and 400× magnification (scale bar = 10 μm) on the right. M-SHRSP, malignant stroke-prone spontaneously hypertensive rats; TCM, trichloromethiazide; WKY, Wistar–Kyoto.
DNA microarray findings
Gene selection criteria
Although initially genes with expression levels more than twice or less than half post-TCM administration were selected, the resulting number was too large for meaningful analysis. Therefore, a stricter criterion was applied, selecting genes expressed more than four-fold or less than 0.25-fold to identify significant genes related to TCM administration.
Six-week-old malignant stroke-prone spontaneously hypertensive rats
At 6 weeks, 58 genes were identified with over four-fold expression, and 34 genes with under 0.25-fold expression. Among these, 13 genes implicated in tissue protection, antioxidant activity, and blood flow regulation were selected (Table 1). Information sources such as GenBank (https://www.ncbi.nlm.nih.gov/gene), Vector builder (https://www.vectorbuilder.jp), and the National BioResource Project for the Rat in Japan (https://www.anim.med.kyoto-u.ac.jp/nbr/defaultöm) were used for gene identification.
TABLE 1.
Considerable messenger RNA expressions increased or decreased by trichloromethiazide administration in the mesenteric artery of malignant stroke-prone spontaneously hypertensive rats
| At 6 weeks of age in the mesenteric artery | ||
| (a) Increasing genes | ||
| (13 of considerable genes/58 genes over four folds/321 genes over two folds expression) | ||
| Gene symbols | Protein name | Fold changes (3% TCM/control) |
| 1. Hspa1a | Heat shock 70 kDa protein 1A | 12.77 |
| 2. Gfap | Glial fibrillary acidic protein | 12.69 |
| 3. Angpt2 | Angiopoietin 2 | 7.68 |
| 4. Mbp | Myelin basic protein | 7.17 |
| 5. Atg16l1 | Autophagy related 16-like 1 | 5.61 |
| 6. Irak2 | Interleukin-1 receptor-associated kinase 2 | 5.11 |
| 7. Inppl1 | Inositol polyphosphate phosphatase-like 1 | 5.05 |
| 8. Aggf1 | Angiogenic factor with G patch and FHA domains 1 | 4.99 |
| 9. Scarb1 | Scavenger receptor class B, member 1 | 4.86 |
| 10. Gnl1 | Guanine nucleotide binding protein-like 1 | 4.76 |
| 11. Prkcq | Protein kinase C, theta, transcript variant 2 | 4.65 |
| 12.Bfar | Bifunctional apoptosis regulator | 4.22 |
| 13. Il6st | Interleukin 6 signal transducer | 4.05 |
| (b) Decreasing genes | ||
| (Six of considerable genes/34 genes under 0.25 folds/132 genes under 0.5 folds expression) | ||
| 1. Col4a1 | Procollagen, typeIV, alpha 1 | 0.024 |
| 2. Myo1a(Bbmi) | Brushborder myosin I | 0.052 |
| 3. Ndufe2 | NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 2 | 0.066 |
| 4. Prpg2 | Proline-rich proteoglycan 2 | 0.139 |
| 5. Atp2b2 | ATPase, Ca++ transporting, plasma membrane 2 | 0.192 |
| 6. Ptgs2 | Prostaglandin-endoperoxide synthase 2 | 0.213 |
| At 9 weeks of age in the mesenteric artery | ||
| (a) Increasing genes | ||
| (One of considerable genes/3 genes over 4.0 folds/4 genes over 2.0 folds expression) | ||
| 1. PI15 | Protease inhibitor 15 | 4.03 |
| (b) Decreasing genes | ||
| (Two of considerable genes /31 genes under 0.25 folds / 59 genes under 0.5 folds expression) | ||
| 1. Oprd1 | Opioid receptor, delta 1 | 0.213 |
| 2. Tnni2 | Troponin I type 2 | 0.223 |
Three percentage of TCM mixed in the chow was administered to M-SHRSP for 2 weeks at 6-week-old and 5 weeks at 9-week-old, respectively.
Fold change was calculated by dividing the amount of mRNA expressed in TCM-administered rats by the amount of mRNA expressed in nonadministered rats. The number of samples in each group was three cases. M-SHRSP, malignant stroke-prone spontaneously hypertensive rats; TCM, trichloromethiazide.
Nine-week-old malignant stroke-prone spontaneously hypertensive rats
At 9 weeks, 3 genes with high expression and 31 genes with low expression were selected, identifying one and two considerable genes, respectively.
Mesenteric artery
The number of significantly upregulated and downregulated mRNAs was lower at 9 weeks compared with 6 weeks, indicating a diminished effect of TCM on mRNA expression in older rats.
Kidneys
In the kidneys, the number of significantly expressed mRNAs did not differ between 6 and 9 weeks of age. At 6 weeks, 68 genes were highly expressed, with 15 showing increased expression and five genes showed low expression, with three being significant (Table 2). At 9 weeks, 27 genes were highly expressed, with 12 identified as significant (Table 2a). Among the 40 downregulated genes, three were considerable (Table 2b).
TABLE 2.
Considerable messenger RNA expressions increased or decreased by trichloromethiazide administration in the kidney of malignant stroke-prone spontaneously hypertensive rats
| At 6 weeks of age in the kidney | ||
| (a) Increasing genes | ||
| (15 of considerable genes/68 genes over 4 folds/261 genes over 2.0 folds expression) | ||
| Gene symbol | Protein name | Fold changes (3.0% TCM/control) |
| 1. Ptgs2 | Prostaglandin-endoperoxide synthase | 11.1 |
| 2. Cfd | Complement factor D (adipsin) | 8.41 |
| 3. Acvr2a | Activin A receptor type 2A | 6.47 |
| 4. Atp12a | ATPase H+/K+ transporting nongastric alpha2 subunit | 6.14 |
| 5. Agtpbp1 | ATP/GTP binding carboxypeptidase 1 | 5.70 |
| 6. Ascc3 | Activating signal cointegrator 1 complex subunit 3 | 5.70 |
| 7. Gup1 | Glycerol uptake/transporter homolog | 5.51 |
| 8. Ren1 | Renin 1 | 5.51 |
| 9. Ptprj | Protein tyrosine phosphatase, receptor type, J | 5.14 |
| 10. Atad2 | ATPase family, AAA domain containing 2 | 5.13 |
| 11. Plcb1 | Phospholipase C, beta 1 | 5.09 |
| 12. Srrm1 | Serine/arginine repetitive matrix 1 | 5.09 |
| 13. Usp24 | Ubiquitin specific protease 24 | 5.05 |
| 14. Lama2 | Laminin, alpha 2 | 4.99 |
| 15. Kng1 | Kininogen 1 | 4.41 |
| (b) Decreasing genes | ||
| (Three of considerable genes/5 genes under 0.25/498 genes under 0.5 folds expression) | ||
| 1. Kb15 | Type II keratin | 0.219 |
| 2. M60206 | Vitamin D binding protein gene | 0.231 |
| 3. BX883048 | Major histocompatibility complex class IIa | 0.235 |
| At 9 weeks of age in the kidney | ||
| (a) Increasing genes | ||
| (Twelve of considerable genes/27 genes over 4 folds/512 genes over 2.0 folds expression) | ||
| 1. Nr1d1 | Nuclear receptor subfamily 1, group D, member 1 | 7.50 |
| 2. Klk3 | Kallikrein, submaxillary gland S3 | 6.82 |
| 3. Ptgs2 | Prostaglandin-endoperoxide synthase 2 | 6.25 |
| 4. Tac2 | Tachykinin 2 | 5.03 |
| 5. Ren1 | Renin 1 | 5.01 |
| 6. Gk11 (Klk1b11 | Glandular kallikrein 11 | 5.43 |
| 7. Gup1 | Glycerol uptake/transporter homolog | 5.35 |
| 8. Kng1 | Kininogen 1 | 4.30 |
| 9. Ccl3 | Chemokine (C-C motif) ligand 3 | 4.24 |
| 10. Ccl20 | Chemokine (C-C motif) ligand 20 | 4.23 |
| 11. Flrt3 | Fibronectin leucine rich transmembrane protein 3 | 4.20 |
| 12. Usp18 | Ubiquitin specific peptidase 18 | 4.09 |
| (b) Decreasing genes | ||
| (Three of considerable genes/40 genes under 0.25/498 genes under 0.5 folds expression) | ||
| 1. Scn4b | Sodium channel, voltage-gated, type IV, beta | 0.234 |
| 2. Ntrk3 | Neurotrophic tyrosine kinase, receptor, type 3 | 0.245 |
| 3.Abra | Actin-binding Rho activating protein | 0.247 |
Three percentage of TCM mixed in the chow was administered to M-SHRSP for 2 weeks at 6-week-old and 5 weeks at 9-week-old, respectively. Fold change was calculated by dividing the amount of mRNA expressed in TCM-administered rats by the amount of mRNA expressed in nonadministered rats. The number of samples in each group was three cases. M-SHRSP, malignant stroke-prone spontaneously hypertensive rats; TCM, trichloromethiazide.
DISCUSSION
The findings from the animal experiments yielded several important results. Initially, SBP in the untreated M-SHRSP group increased with age, reaching levels 11–17.7% higher than those in the 3% TCM group until 10 weeks of age. SBP values in the 0.3% TCM group did not differ significantly from those in the untreated group. After 10 weeks, the control group began to experience stroke-related deaths, causing their SBP to plateau, as shown in Fig. 1. High-dose TCM significantly reduced SBP, but the low-dose TCM group did not show this effect. In the untreated group, rats began to die after 10 weeks, and all rats had died by 14 weeks, as shown in Fig. 2. In contrast, the low-dose TCM group had a survival rate of 54%, whereas the high-dose TCM group survived until 16 weeks. Although the body weights were maintained with no difference among the three groups during 4–10 weeks of age, the weights of the control group were significantly lower than those of the 3% TCM group at 12 weeks of age, likely due to persistent hypertension-related disorders. The organ weights of rats treated with 0.3 and 3% TCM per 100 g body weight were significantly lower in all organs, except for the heart weight of 0.3% TCM group as mentioned in RESULTS, compared with the untreated control group (Fig. 3). This suggests that TCM helps prevent tissue swelling associated with inflammatory changes because of hypertension or stroke. Additionally, plasma TBARS levels, an index of oxidative stress, were significantly lower in both TCM groups compared with the control group, indicating that TCM reduces free radical production and maintains tissue integrity through its antioxidant effects. Histological analysis of kidney tissues stained with H&E and EVG showed that TCM effectively protected against hypertensive tissue disorders such as atherosclerosis, fibrosis, and inflammation. Similar protective effects were observed in the brain and heart tissues following TCM treatment. In the high-dose TCM group, all the animals survived with a decrease in blood pressure until 16 weeks of age, and the kidney, brain, and heart tissues showed any severe organ histological disorders at 12 weeks of age. In addition, the number of rats that died from stroke was significantly less than 20% at 14 weeks of age in the low-dose TCM group even without lowering blood pressure. This indicates that TCM prevented the onset of stroke at low doses without causing hypotensive effects. These results reveal that thiazides prevented organ damage not only through their antihypertensive effects but also by modulating mRNA expression as explaining later.
Selection of involvement genes related with trichloromethiazide giving to malignant stroke-prone spontaneously hypertensive rats in DNA microarray experiments
As both the numbers of increasingly and decreasingly expressed mRNAs were fewer at 9-week-old of age than those at 6-week-old of age in the mesenteric artery, the effects of TCM on mRNA expression would be more pronounced in younger rats than in older rats.
From the 6-week-old mesenteric arteries, several genes with significant changes in expression were identified. Hspa1a (heat shock 70 kDa protein 1A), which stabilizes existing proteins against aggregation, and Atgpt2, part of a large protein complex necessary for autophagy, showed increased expression. Aggf1, an angiogenic factor that promotes of endothelial cell proliferation, and Bfar, involved in the negative regulation of the IRE1-mediated unfolded protein response, were also upregulated. Conversely, Col4a1, an integral component of basement membranes, Atp2b2, critical for intracellular calcium homeostasis, and Ptgs2, a key enzyme in prostaglandin biosynthesis involved in inflammation and mitogenesis, showed decreased expression. At 9 weeks of age, one gene involved in the effects of TCM in the mesenteric artery of M-SHRSP was selected.
In the kidneys of 6-week-old M-SHRSP rats, several genes showed increased expression. These included Ptgs2, Ascc3 (a helicase involved in DNA repair and resistance to alkylation damage), Gup1 (involved in remodeling GPI anchors and misfolded protein quality control), Ptprj (a member of the PTP family regulating various cellular processes), Atad2 (an AAA family protein assisting in protein complex assembly or disassembly), Plcb1 (which catalyzes the formation of 1,4,5-ITP and diacylglycerol), Srrm1 (enabling RNA binding activity involved in mRNA splicing), Usp24 (related to ubiquitin modification), and Lama2 (an extracellular protein in the basement membrane). No genes involved in downregulation were detected in the kidneys.
In 9-week-old kidneys, genes such as Nr1d1 (a transcription factor that negatively regulates core clock protein expression), Ptgs2, Gup1, Flrt3 (a member of the fibronectin leucine-rich transmembrane protein family), and Usp18 (a ubiquitin-specific protease) showed increased expression. The genes Ntrk3 (involved in cell differentiation) and Abra (expressed during arteriogenesis and collateral growth) showed decreased expression.
Based on these findings, Hspa1a, Angpt2, and Bfar were identified as genes related to cell and tissue protection and repair. Aggf1, Col4a1, Atp2b2, and Ptgs2 were identified as genes related to tissue proliferation, maintenance, and function from the mesenteric artery data of 6-week-old M-SHRSPs. From the 6-week-old kidney data, Ascc3, Gup1, Atad2, and Usp24 were identified as genes related to cell and tissue protection and repair, whereas Ptgs2, Ptprj, Plcb1, Srrm1, and Lama2 were associated with tissue proliferation, maintenance, and function. From the 9-week-old kidney data, Gup1, and Usp18 were identified as genes related to cell and tissue protection and repair, whereas Nrld1, Flrt3, Ptgs2, Ntrk3, and Abra were related to tissue proliferation, maintenance, and function. Notably, Ptgs2, Gup1, and Usp24 or Usp18 were commonly expressed across tissues and age groups.
Although no genes identified in this study were directly associated with hypotensive or anti-inflammatory effects, including antioxidant effects, several genes related to tissue protection, repair, and function were discovered in this hypertensive animal model treated with TCM. The antioxidant activity of TCM itself likely contributes to its direct anti-inflammatory effects against hypertensive tissue damage. Previous population genetic studies, such as genome-wide association studies, have discovered multiple pivotal polymorphisms and genetic variants susceptibility to primary hypertension [14–16]. These studies have revealed genetic background and syndromic characteristics of primary hypertension. The present study indicates that some genes not directly related to hypertension also contribute to the pathogenesis of hypertension complications.
A systematic review and meta-analysis by Reinhart et al.[8] in 2023 on hypertension treatment in humans found that thiazides and thiazide-like drugs reduced some morbidity outcomes, such as cardiovascular events, compared with other drugs, despite the absence of a clearly proven mechanism [17–19]. In pharmacological studies, Uehara et al.[20] in 1990 demonstrated that indapamide diuretics increased prostacyclin generation in vascular smooth muscle cells, possibly through antioxidant effects, contributing to its vasodilatory actions. Pourafshar et al.[21] in 2018 reported that thiazides remain effective in individuals with low glomerular filtration rate (GFR), suggesting simultaneous renal and extrarenal mechanisms. Rapoport and Soleimani [22] in 2019 proposed that thiazide chronically reduce arterial pressure through vascular dilation. Additionally, Vergely et al.[23] in 1998 showed that indapamide and 5-OH indapamide were effective superoxide radical anion scavengers or potent radical scavengers using an electron paramagnetic resonance technique or by measuring the oxygen radical-absorbing capacity. Additionally, Zhu et al.[24] in 2016 showed that several benzothiazine derivatives demonstrate strong antioxidant activity. As thiazides have a clinical and pharmacological advantage compared with other drugs as above, it leaded us to speculate that its antioxidant and other unclear effects at now may be positive effects of thiazides.
The pharmacological effects of thiazides on hypertension in humans, however, remain unclear. This experimental study revealed that even at doses that do not affect blood pressure, thiazide TCM suppresses the onset of stroke in M-SHRSP and prevents damage to important organs such as the kidneys, brain, and heart. Although this study has not yet confirmed the mechanism, it seems likely that TCM exerts its effects through the modulation of mRNA expression and the suppression of free radical production, which are related to the preservation of several important organs. The new results from our animal study using M-SHRSP treated with TCM, a thiazide, would provide insights into how thiazides might function at the mRNA level in hypertension, even though the exact candidate genes and mechanisms are yet to be identified.
In conclusion, genes such as Hspa1a, Angpt2, Bfar, Ascc3, Gup1, Atad2, Usp24, and Usp18 are associated with cell and tissue protection and repair. In contrast, Aggf1, Col4a1, Atp2b2, Ptgs2, Ptprj, Plcb1, Srrm1, Lama2, Nrld1, Flrt3, Ntrk3, and Abra are linked to tissue proliferation, maintenance, and function in hypertension treated with TCM. Additionally, the antioxidant properties contribute to its direct anti-inflammatory effects against hypertensive tissue damage.
Future research should focus on elucidating the specific mechanisms by which TCM exerts its antihypertensive and anti-inflammatory effects at the molecular level. Additionally, identifying the precise candidate genes and pathways involved in these processes will enhance our understanding of thiazide pharmacodynamics and potentially lead to more effective hypertension treatments.
ACKNOWLEDGEMENTS
We would like to thank our laboratory staffs for helping our experiment, and Editage (www.editage.jp) for English language editing. This work was supported by the National Center for Biotechnology Information (NCBI), DNA Data Bank of Japan, and the National Bio Resource Project for Rats in Japan for access to their network servers.
Conflicts of interest
There are no conflicts of interest.
Footnotes
Abbreviations: EVG, Elastica-von Gieson; H&E, hematoxylin and eosin; mRNA, messenger RNA; M-SHRSP, malignant type of stroke-prone spontaneously hypertensive rats; TBARS, thiobarbituric acid reactive substances; TCM, trichlormethiazide
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