Role of Calcium Channel Blockers and Diuretics in Salt -induced Oxidative Stress associated Erythrocyte Osmotic Fragility of Male Wistar rats

Role of Calcium Channel Blockers and Diuretics in Salt -induced Oxidative Stress associated Erythrocyte Osmotic Fragility of Male Wistar rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Role of Calcium Channel Blockers and Diuretics in Salt -induced Oxidative Stress associated Erythrocyte Osmotic Fragility of Male Wistar rats Henrietha Nwakaego Chukwuefe, Ubong Edem David, Abodunrin Adebayo Ojetola, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6167252/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background The prevalence of hypertension is rising globally, and elevated blood pressure (BP) is the leading cause of death, with high salt intake as the most common cause for the development of hypertension. Amlodipine is a calcium channel blocker which is effective in the treatment of hypertension and in the prophylactic management of angina while Indapamide is a thiazide-like class of diuretics that reduces blood volume by acting on the kidneys to reduce sodium (Na + ) reabsorption in the distal convoluted tubule. Methods Twenty-four (24) male Wistar rats weighing between 185- 200g were used for this study. The rats were randomly assigned into 4 groups (n = 6). Group one (1) were the Normal control. Group 2: sodium supplemented diet only. Group 3 and 4: Sodium supplemented fed for 8 weeks + Amlodipine (5mg/kgb.w) treatment for four (4) weeks and sodium supplemented fed for 8 weeks + Indapamide (1mg/kgb.w) treatment for 4 weeks. After the fourth week of treatment, the animals were euthanized under ketamine (50mg/kg) anesthesia, and blood sample collected via cardiac puncture into eppendorf bottle to obtain serum for biochemical analysis and lithium heparin bottles for RBC osmotic fragility test. The left ventricle, ascending aorta and kidney were extracted for histological examination. Results Malondialdehyde (MDA) level was significantly increased in Sodium fed rats when compared with control. Amlodipine and Indapamide significantly reduced MDA level in Sodium fed rats. Antioxidant enzymes were also significantly decreased in Sodium fed but was increased by Amlodipine and Indapamide respectively. The sodium fed rats exhibited significantly higher erythrocyte osmotic fragility than the control, sodium fed + Amlodipine and sodium fed + Indapamide groups at 0.3, 0.5 and 0.7% of NaCl concentration. Both systolic and diastolic blood pressures were also significantly increased in sodium fed rats when compared with control but was decreased with Amlodipine and Indapamide treatment. Conclusion Amlodipine and Indapamide demonstrated antioxidant property and ameliorative role on Wistar rats fed with high salt diet. Hypertension Sodium supplemented diet Amlodipine Indapamide antioxidants Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Hypertension, often known as the “silent killer” is a global public health challenge affecting 15–20% of all adults. 1 Hypertension is considered a major risk factor for cardiovascular diseases, and the global prevalence of hypertension and other non-communicable diseases (NCDs) is rapidly increasing, and the African continent seems to be the most affected region in the world, with Nigeria constituting approximately 25% of emergency medical admissions in urban hospitals as the most frequently diagnosed cardiovascular disorder. 2 Although the etiology of hypertension is multifactorial, this includes humoral mediators, vascular reactivity, circulating blood volume, vascular caliber, blood viscosity, cardiac output, blood vessel elasticity and neural stimulation modulate the blood pressure (BP) aside genetic factors and several behavioural, socioeconomic and metabolic factors such as obesity, diabetes and raised blood lipids that acts as risk factors. 3 Environmental factors such as high dietary sodium intake, excess alcohol consumption, smoking, stress, and increased body weight are also risk factors. Among the several mechanisms of elevated blood pressure in response to salt load include enhanced constrictor response of vascular smooth muscle to agonists as well as reduced vasodilatory responses in perfused rat mesenteric artery, changes in vascular reactivity, calcium mobilization, Na+, K+-ATPase activity and endogenous sodium pump ligands levels. 4 A high sodium intake might be related to an increment in vascular reactivity that leads to an increased peripheral vascular resistance, thus producing an impaired endothelium dependent relaxation, increased generation of reactive oxygen species and a down-regulation of nitric oxide synthase (NOS) subtypes. 5 Increase in the generation of reactive oxygen species and decreased antioxidant activities have been shown to be one of the mechanisms of the pathogenesis of hypertension. 6 Free radicals cause oxidative stress as they damage proteins, lipids, carbohydrates, enzyme and DNA in cells and in tissues, thus contributing to the development of cardiovascular diseases, diabetes, autoimmune disorders, neurodegenerative diseases and cancer via DNA fragmentation, lipid peroxidation and membrane damage. Studies have shown that a high salt intake can cause increased pressure on the vascular walls and membranes of red blood cells (RBCs) leading a higher erythrocyte Osmotic fragility (OF). 7 RBCs are highly susceptible to oxidative damage due to the high concentration of oxygen and hemoglobin; it is considered as a powerful promoter of the oxidative process. Oxidative stress and inflammation are cooperative events involved in the pathogenesis of hypertension. Thus, the osmotic fragility might reflect structural and functional abnormalities of cell membranes and could be one of the genetic biomarkers of the hypertensive predisposition. Abnormalities of the physical properties of the membrane and of multiple transport systems have also been implicated in the pathogenesis of hypertension. Antihypertensive treatment helps to reduce not only blood pressure (BP) values but also hypertension-induced organ damage via reduction of oxidative stress. The elevated blood pressure response to high dietary salt intake in rats has been shown to be ameliorated by some agents such as ACE inhibitors like captopril as well as the aldosterone antagonist, spironolactone, 8 implicating the Renin Angiotensin Aldosterone System (RAAS) in the response. Calcium ion Channel Blockers (CCBs) have also been used extensively to treat human hypertension either as monotherapy or in combination therapy. These compounds including the dihydropyridines (e.g. nifedipine and amlodipine) and non-dihydropyridine compounds (e.g. diltiazem and verapamil) lower blood pressure by reducing the entry of calcium ions into the heart and blood vessels However, an earlier report showed that the calcium channel antagonist, nifedipine, does not abolish the high blood pressure response to salt loading in salt diet rats. 9 Amlodipine which is also a member of the dihydropyridine class of calcium antagonists and is believed to have cellular action of selectively inhibiting transmembrane influx of calcium ions into vascular smooth muscle and cardiac muscle, with a greater effect on vascular smooth muscle cells than on cardiac muscle cells. In view of the reported differences in the vascular actions of different CCBs, it is possible that these agents may not be acting solely as vascular calcium ion channel blockers, in reducing blood pressure and that there may be other additional mechanisms involved. Indapamide belongs to the thiazide-like class of diuretics, and it is used as an oral antihypertensive agent where it reduces blood volume by acting on the kidneys to reduce sodium (Na + ) in the distal convoluted tubule. 10 Indapamide also act in the treatment of hypertension by reducing cardiac hypertrophy and thickening of arterial walls, prevents accumulation of the embryonic isoform of fibronectin in coronary vessels, scavenges free radicals, leading to stimulation of vasodilator eicosanoid formation, and interaction with renal carbonic anhydrase. The prevalence of hypertension is rising globally, and high salt intake which can cause excessive production of reactive oxygen species (ROS), overwhelming the antioxidant defense leads to increased pressure on the vascular walls and increased mechanical pressure on the membranes of red blood cells (RBCs) leading a higher erythrocyte Osmotic fragility (OF), which may be the major cause for progression to the development of cardiovascular disease (CVD) 7 and pathogenesis of hypertension. 6 Materials and Methods Experimental Animals Twenty-four (24) Male Wistar rats, weighing between 185–200 g were used for this study. The animals were obtained from Central Animal House, College of Medicine, University of Ibadan. They were acclimatized for 2 weeks and housed in solid bottom polypropylene cages under standard environmental conditions in a well-ventilated room under a condition of room temperature (approximately 23–25 0 C), relative humidity (approximately 55%) and natural environmental light (12/12- hour light/dark cycle) respectively according to the guidelines for the care and use of laboratory animals of the University of Ibadan, Nigeria. The animals were allowed access to standard grower’s feed (Top Feeds, Nigeria) and water ad libitum. The animals were acclimatized for two weeks and thereafter randomly divided into four groups A, B, C and D of 6 rats per group. Group A (control rats) were fed with normal feed. Group B were fed with salt supplemented diet (group B) only for 8 weeks. Group C were fed with salt supplemented feed for 8 weeks and treated with amlodipine for 4 weeks. Group D were fed with salt supplemented feed for 8 weeks and treated with indapamide for 4 weeks. Preparation and Administration of Drugs and Chemicals Norvasc Amlodipine and Indapamide were purchased from a local pharmaceutical store in Ibadan, Nigeria. 5mg/kg body weight of Amlodipine, 11 and 1mg/kg body weight for Indapamide 12 was administered once daily for four weeks. A sodium loaded diet containing 8% NaCl was prepared by adding appropriate quantities of salt to the standard rat feed containing 0.3% NaCl (supplied by Top feeds, Ibadan). 4 Sample Collection On the 28th day after drug administration, all animals were euthanized under ketamine (50mg/kg) anesthesia and blood sample were collected via cardiac puncture into Eppendorf tubes for oxidative biomarkers, 13 and into lithium heparinized tubes for hematological studies (erythrocyte osmotic fragility). The left ventricle, ascending aorta and kidney were extracted for histological examination. Biochemical Analysis Malondialdehyde (MDA) which is an end product of lipid peroxidation is determined by measuring the thiobarbituric acid reactive substances (TBARS) produced during lipid peroxidation according to the method described by Varshney and Kale. 14 Reduced glutathione level (GSH) was estimated by following the protocol of Beutler et al. 15 . The level of SOD activity was determined by the method of Fridovich and Misra. 16 Catalase activity was determined according to the method of Sinha. 17 Nitric Oxide (NO) levels were measured as total nitrite plus nitrate levels using the Griess reagent as described by Moshage et al . 18 Erythrocyte Osmotic Fragility Erythrocyte osmotic fragility was determined based on the modified method by Oyewale et al. 19 Blood from each rat was collected into heparinized sample bottles. Serial solution of sodium chloride (pH 7.4) was prepared in the following concentrations 0.0, 0.1, 0.3, 0.5, 0.7, 0.9 g/d. 1ml of the heparinized blood was pipette into the serial solutions of sodium chloride and mixed carefully. The solution was incubated for 30 minutes at room temperature (18-25 0 C). The test tubes were then centrifuged at 1500 revolutions per minute (rpm) for 10 minutes using a centrifuge. The supernatant was transferred into a glass cuvette after which the concentration of the haemoglobin in the supernatant was measured calorimetrically by reading the absorbance at a wavelength of 540nm using a spectrophotometer. The percentage hemolysis was calculated using the following formula: Percentage hemolysis = Optical density of test solution x 100 Optical density of water Preparation of Tissues for Histology The procedure was carried out according to method of Avwioro, 20 Measurement of Blood Pressure and Heart Rate The systolic and diastolic blood pressures were measured using a CODA non-invasive method (Kent Scientific Co., USA). The CODA tail-cuff system uses Volume Pressure recording (VPR) with specially designed differential pressure transducer to measure the blood pressure in the tail non-invasively by determining the tail volume. This is done at a temperature of 20 0 C after restraining the rat for at about 10–15 minutes before the recording begins. VPR will actually measure six parameters simultaneously: Systolic blood pressure, Diastolic blood pressure, Mean blood pressure, Heart pulse rate, Tail blood volume and Tail blood flow. Statistical Analysis Statistical analysis was carried out using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Data are expressed as Mean ± SEM. Comparison between the groups were performed by One-way and Two-way ANOVA. P < 0.05 was considered statistically significant. Results There was no significant difference in the relative weight for both the left ventricle and kidney between the control groups and the treated groups. The result shows a significant increase in malondialdehyde level (MDA) of the sodium fed only group when compared with the normal control group in serum (A), left ventricle (B), kidney (C) and ascending aorta (D) respectively. Significant reduction in MDA level in serum, left ventricle and kidney was observed in animals treated with sodium + amlodipine and sodium + indapamide group when compared with the sodium fed only group respectively. Result from Figure 3 above shows that sodium fed only group showed a significant decrease in superoxide dismutase level when compared with the normal control group in serum, left ventricle, kidney and ascending aorta respectively. The group treated with sodium + amlodipine also showed a significant increase in the superoxide dismutase level only in serum, left ventricle and Ascending aorta when compared with the sodium fed only group. There was also a significant increase in the superoxide dismutase level in sodium + indapamide group when compared to the sodium fed only group in serum and ascending aorta respectively. Result from Figure 4 above observed that sodium fed only group showed a significant decrease in glutathione reductase level when compared with the normal control group in serum and ascending aorta respectively. More so, the groups treated with sodium + amlodipine and sodium + indapamide showed a significant decrease (in the ascending aorta) and a significant increase (in the serum) in the glutathione reductase level when compared with the sodium fed only group respectively. Result from Figure 5, reports that sodium fed only group showed a significant decrease in catalase level in the serum and ascending aorta respectively when compared with the normal control group. The group treated with sodium + amlodipine showed a significant increase in the catalase level in the serum and ascending aorta when compared with the sodium fed only group. There was also a significant increase in the catalase level in sodium + indapamide group in both the serum and ascending aorta only when compared to the sodium fed only group. Result from Figure 6 above reports that, the sodium fed only group showed only a significant decrease in nitrite level in the left ventricle and kidney when compared with the normal control group. The groups treated with sodium + amlodipine and sodium + indapamide showed a significant decrease (in the left ventricle) and a significant increase (in the kidney) in the nitrite level when compared with the sodium fed only group respectively. Figure 7 shows the effect of Amlodipine and Indapamide on Systolic and Diastolic blood pressures. There was a significant increase in both the Pre-treatment Systolic and diastolic blood pressure in the sodium fed only group when compared with the control group. There was also a significant decrease in the Pre-treatment Systolic and Diastolic blood pressure in the sodium fed + Amlodipine and Sodium Fed + Indapamide group when compared with the Sodium Fed only group. A significant decrease in Post treatment systolic and diastolic blood pressure was recorded in all the treated groups when compared with the Sodium Fed only group. There was a significant difference in the pre-treatment heart rate between the control groups and treated groups. However, a significant decrease in heart rate was recorded in the Post treatment heart rate in sodium fed + Amlodipine and sodium fed + Indapamide when compared with the sodium fed only group. The Sodium fed only group has significantly higher erythrocyte osmotic fragility than the Control, Sodium fed + Amlodipine and Sodium fed + Indapamide groups at 0.3, 0.5 and 0.7% of NaCl concentration, however the erythrocyte osmotic fragility in the Control, Sodium fed + Amlodipine and Sodium fed + Indapamide groups were not significantly different at this concentration. The erythrocyte osmotic fragility in the Control, Sodium fed only, Sodium fed + Amlodipine and Sodium fed + Indapamide groups were not significantly different at 0.9% of NaCl concentration. Figure 10 show a photomicrograph of renal tissue show normal glomeruli, bowman capsule and tubules with no significant lesion seen (A). In Sodium fed rats, severe congestion of the glomeruli and several degeneration of the subcapsular tubules was observed (B). Mild renal cortical congestion was observed in Sodium fed + Amlodipine group (C). Normal glomeruli, bowman capsule and tubules with no significant lesion were observed in Sodim fed + Indapamide group (D). Figure 11 show a photomicrograph of cardiac tissue with normal cytoarchitecture and no significant lesion (A). Mild congestion of the coronary vessels was observed (B). Cardiac tissue shows no significant lesion (C & D). Figure 12 show a photomicrograph of ascending aorta with normal cytoarchitecture and no significant lesion (A). Ascending aorta also shows several tubules with protein casts in their lumina (B). Ascending aorta shows no significant lesion (C & D). Discussion This study evaluated the effects of a Calcium ion Channel Blockers (CCBs)(amlodipine) and diuretics (Indapamide) on changes in oxidative stress and erythrocyte osmotic fragility of Wistar rats fed with high salt diet (8% NaCl) to induce hypertension. The results of the present study confirmed previous studies that in Wistar rats, ingestion of a high salt diet resulted in hypertension. 4 Excessive production of reactive oxygen species (ROS) has been associated with a high salt intake which overwhelms the antioxidant defense and increases pressure on the vascular wall, thus increasing mechanical pressure on the membranes of red blood cells (RBCs) leading to a higher erythrocyte Osmotic fragility (OF), which may be the major cause for the progression to the development of CVD, 7 and pathogenesis of hypertension. 6 Result from the current study (Fig. 2 ) shows that a high salt diet affects both the enzymatic and non-enzymatic antioxidants as well as causes membrane lipid peroxidation, thus raising malondialdehyde levels, an observation that is consistent with previous reports. 6 – 7 The MDA levels in serum, kidney, ascending aorta and left ventricle in this study were significantly decreased in the sodium fed treated with amlodipine and indapamide when compared with the sodium fed only group. This decrease in MDA could be either a result of blood pressure (BP) reduction or antioxidant property of amlodipine and indapamide. These results are in agreement with various other studies that have shown MDA levels to decrease after amlodipine and indapamide administration. 21 Our result indicates that the superoxide dismutase (SOD) level (Fig. 3 ) in serum, kidney, ascending aorta and left ventricle at four weeks were significantly reduced in the salt-sensitive hypertensive rats (SHR) when compared with normal control group. This could be due to the fact that a high salt diet generates increased production of reactive oxygen species especially superoxide anion. The superoxide radical produced is neutralized by superoxide dismutase and the decrease in the level of this antioxidant reveals that a high salt diet leads to increased oxidative stress. 22 Several studies have also reported a low level of SOD in hypertensive patients. This decrease may also be associated with a decrease in NO levels, reinforcing that SOD is helpful in the release of NO, this corroborates findings from the current study. However, treatment with amlodipine and indapamide significantly increased the SOD level of these experimental rats when compared with the sodium fed only group. These findings are in agreement with various other studies that have shown SOD levels to increase after antihypertensive treatment. 23 Glutathione levels in the serum, kidney, ascending aorta and left ventricle were also significantly reduced in the SHR rats when compared with the normal control group (Fig. 4 ). The level of reduced glutathione is a measure of the cellular redox status, hence alteration in glutathione level may affect the overall redox status of the cell. Aniya and Naito, 24 had reported that severe oxidative stress might result in decrease in glutathione s-transferase as well as glutathione. This further confirms that there is an appreciable level of oxidative stress in the group fed with a high salt diet. The glutathione reductase levels were significantly increased in serum in this study (Fig. 4 A) in the sodium fed rats treated with amlodipine and indapamide groups when compared with the sodium fed only group. Catalase is usually inactivated by hydrogen peroxide and superoxide radicals. Hence, reduction in residual catalase level confers increased susceptibility of the cell to undergo lipid peroxidation. This increase in lipid peroxidation is in agreement with the study carried out on hypertensive patients. 25 Our result further indicated that a high salt diet significantly decreased catalase activity in serum, kidney, ascending aorta and left ventricle when compared to the normal control group (Fig. 5 ). This could be due to some unknown mechanism in the oxidative stress process that has affected its activity. It is generally believed that hydrogen peroxide can be detoxified and removed by catalase at high concentration and glutathione peroxidase can destroys it when present at a steady state. 26 The reduction in the level of this enzyme may render the kidney more susceptible to hydrogen peroxide induced oxidative stress. The catalase levels in serum and ascending aorta in this study were significantly increased in the sodium fed groups treated with amlodipine and indapamide when compared with the sodium fed only group, which clearly indicate that amlodipine and indapamide have antioxidative properties, and thus possible renoprotective properties which might be conferred through the reduction or elimination of oxidative stress in the kidney as has been shown for other antioxidants. 27 . Results from present study also showed that nitrite levels in the serum, kidney, ascending aorta and left ventricle were significantly reduced in the SHR rats when compared with the normal control group (Fig. 6 ). Several studies have reported a low level of nitrite in hypertensive patients due to an increased generation of reactive oxygen species and a down-regulation of nitric oxide synthase (NOS) subtypes. 5 Reactive oxygen species may act through several mechanisms to mediate vascular change in hypertension: direct actions on endothelial cells; increase quenching of nitric oxide, a vasodilator by O 2 -.The nitrite levels in left ventricle in this study were also further significantly decreased in the sodium fed treated with amlodipine and indapamide groups respectively when compared with the sodium fed only group. However, the nitrite level in the kidney increased in the sodium fed treated with amlodipine and indapamide groups respectively. Although the impact of different types of antihypertensive treatment on oxidative stress has been analyzed in several studies, results showed reduction in oxidative stress and an increase in antioxidant mechanisms after different antihypertensive treatments. 28 In the current study, the blood pressure of the rats was measured before they were placed on treatment with Amlodipine and Indapamide. The result shows a significant increase in the Pre-treatment systolic and diastolic blood pressure in the sodium fed only group when compared with normal control group (Fig. 7 ), which is consistent with previous research that high sodium intake has a tendency of increasing systolic blood pressure. A significant decrease was observed in the post-treatment systolic and diastolic blood pressure of Amlodipine and Indapamide treatment groups when compared with the sodium fed only group. A significant decrease in heart rate was recorded in the post-treatment heart rate in the sodium fed treated with amlodipine group and the sodium fed treated with indapamide group when compared with sodium fed only group (Fig. 8 ). Red blood cells (RBCs) being the most abundant cells in humans are the first cells to be prone to stressful stimuli, and because of their role as O 2 and CO 2 transporter, they are under constant exposure to reactive oxygen species (ROS) and oxidative stress. 29 The results of the present study showed a significantly increased erythrocyte osmotic fragility (EOF) at 0.3, 0.5 and 0.7% of NaCl concentrations in the sodium fed only group compared to normal control group and sodium + amlodipine treated group as well as the sodium + indapamide treated group (Fig. 9 ). This result could be due to the pro-action of high salt diet on ROS level thereby resulting in lipid peroxidation of the membrane lipid bi-layers which are rich in polyunsaturated fatty acids side chains. The lipid peroxidative alteration in the structural and functional components of the erythrocyte membrane may have caused perturbations in the membrane integrity resulting in increased erythrocyte fragility recorded in the sodium fed only group in the present study. The increased EOF observed indicates the ability of the high salt diet to compromise the integrity of the erythrocyte membrane apparently from increased oxidative damage. 7 Our results are in agreement with previous studies that high salt diet induces oxidative stress, via the formation of free radicals and reactive oxygen species (ROS), thus increasing osmotic fragility of RBCs. 7 The decrease observed in the sodium + amlodipine and sodium + indapamide treated groups could have been from the antioxidant activity of the antihypertensive therapy, which enables them to mop up the prevalent reactive oxygen species, resulting in less damage to the cell membrane of the erythrocytes. This result is in agreement with Fasanmade. 30 who investigated the beneficial effects of diuretic therapy on erythrocyte osmotic fragility in hypertension during diuretic therapy and found out that erythrocyte osmotic fragility was decreased in the group that received diuretic therapy. Fujita et al. 31 also demonstrated the ability of calcium channel blockers to offer protective effect on RBC deformability. The reduction of the EOF by antihypertensive therapy observed in the sodium + amlodipine and sodium + indapamide treated groups further confirms the role of oxidative stress in the toxic mechanism of erythrocyte damage observed in the sodium fed only group in the present study. The ability of calcium channel blockers to preserve cellular membrane integrity through its capacity to exhibit membrane modulatory effects may be responsible for this ameliorative effect. 31 Histological studies reveal congestion of the interstitium of the kidney with several tubules, especially the subcapsular ones severely degenerated along with a moderate congestion of the glomeruli and several interstitial vessels in the sodium fed only group which was restored towards normal in the groups receiving the 8% NaCl diet with Amlodipine and Indapamide treatment (Fig. 10 – 12 ). This finding aligns with results obtained from previous studies in which male Wistar albino rats receiving 8% NaCl diet for 8 weeks displayed considerable degrees of tubular degeneration and renal tissue congestion. 32 However, cardiac tissue showed mild congestion of the coronary vessels in the sodium fed only group which was restored towards normal in the groups receiving the 8% NaCl diet with Amlodipine and Indapamide treatment. This observation could be due to increased blood pressure observed in the groups fed on a high salt diet, as the elevated vascular resistance observed in hypertension has been linked to structural changes in the blood vessel (Gros et al., 2000). This finding corroborates the histoarchitectural phenotype of the coronary vessels of the rats on high-salt diet as reported by Fadahunsi and Olorunnisola. 33 The photomicrograph of ascending aorta showed several tubules have protein casts in their lumina in the sodium fed only group which was also restored towards normal in the groups receiving the 8% NaCl diet with Amlodipine and Indapamide treatment. The protein casts in the lumina of the aortic section may result into thickening of the wall of the aorta. 34 Our findings are consistent with the reported histoarchitectural changes of the aorta of the rats on high-salt diet. 35 Conclusion This study suggests that Amlodipine and Indapamide have an ameliorative effect on oxidative stress level of Wistar rats fed with high sodium diet. Wistar rats fed with high sodium supplemented food tend to have high blood pressure. This study also confirms the blood pressuring lowering and antioxidant effects of amlodipine, with Indapamide having a higher potency in reducing blood pressure via increasing sodium excretion. Abbreviations Na + sodium ROS reactive oxygen species SHR salt-sensitive hypertensive rats RBCs red blood cells BP blood pressure NOS nitric oxide synthase CCBs calcium channel blockers SOD superoxide dismutase SOD CAT catalase GSH reduced glutathione level MDA malondialdehyde EOF erythrocyte osmotic fragility SF Only Sodium Fed SF + AMD Sodium Fed and Amlodipine SF + IND Sodium Fed and Indapamide NCDs non-communicable diseases NOS nitric oxide synthase NO nitric oxide NaCl sodium chloride CVD cardiovascular diseases RAAS renin angiotensin aldosterone system Declarations Ethics approval and consent to participate Ethical approval was obtained from the Animal Care and Use Research Ethics Committee of the University of Ibadan, Nigeria. Informed consent: Informed consent was obtained from all individuals included in this study. Consent for publication All authors have read and agreed to the publishing of the manuscript. Availability of data and materials Data will be made available on request. Declaration of competing interest Authors declare that they have no conflict of interest. Funding This research received no specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author’s contribution Henrietha Nwakaego Chukwuefe: Writing – original draft, Investigation, Funding acquisition, Methodology, Formal analysis, Data curation, Conceptualization. Ubong Edem David: Validation, Formal analysis, Data curation. Abodunrin Adebayo Ojetola: Writing – review & editing. Adesoji Adedipe Fasanmade: Validation, Supervision, Project administration, Conceptualization. Acknowledgement Not applicable References Marshall IJ, Wolfe CD, McKevitt C. Lay perspectives on hypertension and drug adherence. 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Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity. 2014. http://dx.doi.org/10.1155/2014/608512 Aniya Y and Naito A. Oxidative stress-induced activation of microsomal glutathione S-transferase with glutathione peroxidase, Gene Ther 1993, 9(2):110-117. Manuela G, Xuejuan J, and Esteban Castelao J. Lipid peroxidation, oxidative stress genes and dietary factors in breast cancer protection: a hypothesis. Breast Cancer Res 2007, 9(1):201. Casado A, Dela Torre R, Lopez-Fernandez M. et al. catalase is associated with blood pressure levels", Hum.Genet 1995, 109(1)95-98. Tian N, Thrasher KD, Gundy PD. et al. Antioxidant treatment prevents renal damage and dysfunction and reduces arterial pressure in salt-sensitive hypertension,” Hypertension , 2005, 45 (5)934–939. Ghiadoni L, Magagna A, Versari D. et al., Different effect of antihypertensive drugs on conduit artery endothelial function. Hypertension 2003, 41:1281–1286. Pandey KB. and Rizvi SI. Markers of oxidative stress in erythrocytes and plasma during aging in humans. Oxid Med Cell Longev 2010, 3(1): 2-12. Fasanmade AA. Erythrocyte osmotic fragility in hypertension and during diuretic therapy. West Afr J Med 1999;18(3):183-186. Fujita J, Tsuda K, Takeda T. et al. Nisoldipine improves the impaired erythrocyte deformability correlating with elevated intracellular free calcium-ion concentration and poor glycaemic control in NIDDM. British Journal of Clinical Pharmacology 1999, 47(5):499-506. ⁠Khadive T, Ghadimi D, Hemmati M, Golshahi H. Impact of high salt diets on CHOP-mediated apoptosis and renal fibrosis in a rat model. Mol Biol Rep 2021, 48: 6423–6433 Fadahunsi OS, Olorunnisola OS. High-salt–fat diet: a risk factor for elevated blood pressure associated with dyslipidemia, perturbation in cardio-renal anti-oxidant and pro-inflammatory status in Wistar rats. JoBAZ 2024, 85: 62. ⁠Marelli AJ, Perloff JK, Child JS. Et al. Pulmonary atresia with ventricular septal defect in adults. Circulation . 1994; 89(1):243-51. Akukwu D, Ugochukwu A, Catherine A. et al. Effect of High Salt Diet and Habiscus Sabdariffa (Zobo) on the Histology of the Aorta in Wistar Rats. International Research Journal of Gastroenterology and Hepatology 2023, 6 (1):108-20. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 27 May, 2025 Reviewers agreed at journal 18 May, 2025 Reviewers invited by journal 30 Apr, 2025 Editor assigned by journal 25 Apr, 2025 Editor invited by journal 31 Mar, 2025 Submission checks completed at journal 28 Mar, 2025 First submitted to journal 28 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6167252","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":451133828,"identity":"7926aad2-03f9-45c2-a4a2-f77a0b553ccb","order_by":0,"name":"Henrietha Nwakaego Chukwuefe","email":"data:image/png;base64,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","orcid":"","institution":"University of Ibadan","correspondingAuthor":true,"prefix":"","firstName":"Henrietha","middleName":"Nwakaego","lastName":"Chukwuefe","suffix":""},{"id":451133829,"identity":"66b0b2e2-61f7-4c5a-9578-c536258665bf","order_by":1,"name":"Ubong Edem David","email":"","orcid":"","institution":"Ajayi Crowther University","correspondingAuthor":false,"prefix":"","firstName":"Ubong","middleName":"Edem","lastName":"David","suffix":""},{"id":451133830,"identity":"ab64e37b-eeb6-4785-bda2-d1d43b0e3487","order_by":2,"name":"Abodunrin Adebayo Ojetola","email":"","orcid":"","institution":"Adeleke University","correspondingAuthor":false,"prefix":"","firstName":"Abodunrin","middleName":"Adebayo","lastName":"Ojetola","suffix":""},{"id":451133831,"identity":"aa89ce49-fe0c-44e2-81e6-bb34f59e6729","order_by":3,"name":"Adesoji Adedipe Fasanmade","email":"","orcid":"","institution":"University of Ibadan","correspondingAuthor":false,"prefix":"","firstName":"Adesoji","middleName":"Adedipe","lastName":"Fasanmade","suffix":""}],"badges":[],"createdAt":"2025-03-06 05:38:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6167252/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6167252/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82006530,"identity":"3208eeb0-4291-45a5-bd6a-2967e026f564","added_by":"auto","created_at":"2025-05-05 22:54:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":102688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Amlodipine and Indapamide on Relative weight of the left ventricle (A) and kidney (B).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eValues are expressed as mean ± SEM, (n=5); Control – Normal Control; SF Only – Sodium Fed Only; SF + AMD – Sodium Fed and Amlodipine; SF + IND – Sodium Fed and Indapamide\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6167252/v1/716dd1b68a79e0e0b786284a.jpg"},{"id":82006492,"identity":"9e590209-a230-4315-a264-3a77dcb6b09e","added_by":"auto","created_at":"2025-05-05 22:54:48","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":101902,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Amlodipine and Indapamide on Serum (A), left ventricle (B), kidney (C) and Ascending aorta (D) Malondialdehyde level (MDA). \u003c/strong\u003e\u003cem\u003eValues are expressed as mean ± SEM, (n=5); Control – Normal Control; SF Only – Sodium Fed Only; SF + AMD – Sodium Fed and Amlodipine; SF + IND – Sodium Fed and Indapamide. **a p \u0026lt; 0.05 is significant when compared with the normal control group. **b p \u0026lt; 0.05 and **c p \u0026lt; 0.05 is significant when compared with the sodium fed only group.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6167252/v1/7ea38d75561ccc5a053d198e.jpg"},{"id":82006494,"identity":"58b99100-9665-4855-bf72-25bfd562789e","added_by":"auto","created_at":"2025-05-05 22:54:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":120162,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Amlodipine and Indapamide on Serum (A), left ventricle (B), kidney (C) and Ascending aorta (D) superoxide dismutase level (SOD). \u003c/strong\u003e\u003cem\u003eValues are expressed as mean ± SEM, (n=5); Control – Normal Control; SF Only – Sodium Fed Only; SF + AMD – Sodium Fed and Amlodipine; SF + IND – Sodium Fed and Indapamide. **a p \u0026lt; 0.05 is significant when compared with the normal control group. **b p \u0026lt; 0.05 and **c p \u0026lt; 0.05 is significant when compared with the sodium fed only group.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6167252/v1/485566be4b38bedcc801a9da.jpg"},{"id":82006529,"identity":"4527adf8-d9f3-4a42-a25d-6813437fcb0e","added_by":"auto","created_at":"2025-05-05 22:54:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":119693,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Amlodipine and Indapamide on Serum (A), left ventricle (B), kidney (C) and Ascending aorta (D) Glutathione reductase (GSH) level. \u003c/strong\u003e\u003cem\u003eValues are expressed as mean ± SEM, (n=5); Control – Normal Control; SF Only – Sodium Fed Only; SF + AMD – Sodium Fed and Amlodipine; SF + IND – Sodium Fed and Indapamide. **a p \u0026lt; 0.05 is significant when compared with the normal control group. **b p \u0026lt; 0.05 and **c p \u0026lt; 0.05 is significant when compared with the sodium fed only group.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6167252/v1/23dffd80638c36512cd19273.jpg"},{"id":82007013,"identity":"5b7677b7-7fb2-40e9-ab5a-b567187d2f1b","added_by":"auto","created_at":"2025-05-05 23:10:48","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":129418,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Amlodipine and Indapamide on Serum (A), left ventricle (B), kidney (C) and Ascending aorta (D) Catalase (CAT) level. \u003c/strong\u003e\u003cem\u003eValues are expressed as mean ± SEM, (n=5); Control – Normal Control; SF Only – Sodium Fed Only; SF + AMD – Sodium Fed and Amlodipine; SF + IND – Sodium Fed and Indapamide. **a p \u0026lt; 0.05 is significant when compared with the normal control group. **b p \u0026lt; 0.05 and **c p \u0026lt; 0.05 is significant when compared with the sodium fed only group.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6167252/v1/32a02012117a8265a46d58e9.jpg"},{"id":82006498,"identity":"e082cc6b-b62c-4878-91a7-bccb8a66e2ae","added_by":"auto","created_at":"2025-05-05 22:54:48","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":97826,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Amlodipine and Indapamide on Serum (A), left ventricle (B), kidney (C) and Ascending aorta (D) Nitrite level. \u003c/strong\u003e\u003cem\u003eValues are expressed as mean ± SEM, (n=5); Control – Normal Control; SF Only – Sodium Fed Only; SF + AMD – Sodium Fed and Amlodipine; SF + IND – Sodium Fed and Indapamide. **a p \u0026lt; 0.05 is significant when compared with the normal control group. **b p \u0026lt; 0.05 and **c p \u0026lt; 0.05 is significant when compared with the sodium fed only group.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6167252/v1/8b7c77c9b9b4ef95f5d8eee4.jpg"},{"id":82006772,"identity":"d865ecd0-533d-419a-bc6e-1acafc515b7c","added_by":"auto","created_at":"2025-05-05 23:02:48","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":102181,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Amlodipine and Indapamide on Systolic (A)and Diastolic Blood Pressures (B). \u003c/strong\u003e\u003cem\u003eValues are expressed as mean ± SEM, (n=5); Control – Normal Control; SF Only – Sodium Fed Only; SF + AMD – Sodium Fed and Amlodipine; SF + IND – Sodium Fed and Indapamide. \u003c/em\u003e***a p \u0026lt; 0.05, ***b \u0026lt; 0.05, and ***c \u0026lt; 0.05 is significant when compared with Normal control group. # p \u0026lt; 0.05 is significant when compared with the Sodium fed only group.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6167252/v1/6fa2aa495d9edc7ccebea047.jpg"},{"id":82006774,"identity":"862a8c20-c37e-4552-8e70-2dda2dc4c632","added_by":"auto","created_at":"2025-05-05 23:02:48","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":69958,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Amlodipine and Indapamide on Heart rate. \u003c/strong\u003e\u003cem\u003eValues are expressed as mean ± SEM, (n=5); Control – Normal Control; SF Only – Sodium Fed Only; SF + AMD – Sodium Fed and Amlodipine; SF + IND – Sodium Fed and Indapamide. \u003c/em\u003e***a p \u0026lt; 0.05, ***b \u0026lt; 0.05, and ***c \u0026lt; 0.05 is significant when compared with Normal control group. # p \u0026lt; 0.05 is significant when compared with the Sodium fed only group\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6167252/v1/f22e0af4ef8b755ee98838b3.jpg"},{"id":82006499,"identity":"4deb68b4-9f3d-45ce-9efd-409dd8832428","added_by":"auto","created_at":"2025-05-05 22:54:48","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":46256,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Amlodipine and Indapamide on Erythrocyte Osmotic Fragility.\u003c/strong\u003e Values are expressed as mean ± SEM, n (=5). P value ≤ 0.05. \u003cem\u003eControl – Normal Control; SF Only – Sodium Fed Only; SF + AMD – Sodium Fed and Amlodipine; SF + IND – Sodium Fed and Indapamide.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6167252/v1/af08d81f48d3cff4814050b2.jpg"},{"id":82006532,"identity":"c3992e3e-ece4-4409-8c71-ac105124383c","added_by":"auto","created_at":"2025-05-05 22:54:52","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":163509,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotomicrograph of the effect of Amlodipine and Indapamide onrenal histoarchitecture. Normal control group (A), Sodium Fed Only (B), SF + AMD (C) and SF + IND (D). Magnification X400.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6167252/v1/49240d2a3ec100a09d998ce4.jpg"},{"id":82006770,"identity":"fb5a008e-f108-4ea6-ad4d-ef2a857e8e38","added_by":"auto","created_at":"2025-05-05 23:02:48","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":168032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotomicrograph of the effect of Amlodipine and Indapamide onleft ventricular histoarchitecture. Normal control group (A), Sodium Fed Only (B), SF + AMD (C) and SF + IND (D). Magnification X100 and X400.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6167252/v1/bdbb5cd6de3cd8daf1645b9f.jpg"},{"id":82006503,"identity":"0c96a6ed-6820-4d1b-976c-5fa21a855314","added_by":"auto","created_at":"2025-05-05 22:54:48","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":109766,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotomicrograph of the effect of Amlodipine and Indapamide onascending aorta histoarchitecture. Normal control group (A), Sodium Fed Only (B), SF + AMD (C) and SF + IND (D). Magnification X400.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6167252/v1/53e9b405901b50b90d1fe3af.jpg"},{"id":82007336,"identity":"8de82b6c-6a55-412f-8959-9bd6e527e0f7","added_by":"auto","created_at":"2025-05-05 23:26:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2662697,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6167252/v1/0f43dd85-4374-4ba2-aca4-a16bbb9abf7f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Role of Calcium Channel Blockers and Diuretics in Salt -induced Oxidative Stress associated Erythrocyte Osmotic Fragility of Male Wistar rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHypertension, often known as the \u0026ldquo;silent killer\u0026rdquo; is a global public health challenge affecting 15\u0026ndash;20% of all adults. \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Hypertension is considered a major risk factor for cardiovascular diseases, and the global prevalence of hypertension and other non-communicable diseases (NCDs) is rapidly increasing, and the African continent seems to be the most affected region in the world, with Nigeria constituting approximately 25% of emergency medical admissions in urban hospitals as the most frequently diagnosed cardiovascular disorder.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAlthough the etiology of hypertension is multifactorial, this includes humoral mediators, vascular reactivity, circulating blood volume, vascular caliber, blood viscosity, cardiac output, blood vessel elasticity and neural stimulation modulate the blood pressure (BP) aside genetic factors and several behavioural, socioeconomic and metabolic factors such as obesity, diabetes and raised blood lipids that acts as risk factors.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Environmental factors such as high dietary sodium intake, excess alcohol consumption, smoking, stress, and increased body weight are also risk factors. Among the several mechanisms of elevated blood pressure in response to salt load include enhanced constrictor response of vascular smooth muscle to agonists as well as reduced vasodilatory responses in perfused rat mesenteric artery, changes in vascular reactivity, calcium mobilization, Na+, K+-ATPase activity and endogenous sodium pump ligands levels.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA high sodium intake might be related to an increment in vascular reactivity that leads to an increased peripheral vascular resistance, thus producing an impaired endothelium dependent relaxation, increased generation of reactive oxygen species and a down-regulation of nitric oxide synthase (NOS) subtypes. \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Increase in the generation of reactive oxygen species and decreased antioxidant activities have been shown to be one of the mechanisms of the pathogenesis of hypertension.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Free radicals cause oxidative stress as they damage proteins, lipids, carbohydrates, enzyme and DNA in cells and in tissues, thus contributing to the development of cardiovascular diseases, diabetes, autoimmune disorders, neurodegenerative diseases and cancer via DNA fragmentation, lipid peroxidation and membrane damage. Studies have shown that a high salt intake can cause increased pressure on the vascular walls and membranes of red blood cells (RBCs) leading a higher erythrocyte Osmotic fragility (OF).\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e RBCs are highly susceptible to oxidative damage due to the high concentration of oxygen and hemoglobin; it is considered as a powerful promoter of the oxidative process. Oxidative stress and inflammation are cooperative events involved in the pathogenesis of hypertension. Thus, the osmotic fragility might reflect structural and functional abnormalities of cell membranes and could be one of the genetic biomarkers of the hypertensive predisposition. Abnormalities of the physical properties of the membrane and of multiple transport systems have also been implicated in the pathogenesis of hypertension.\u003c/p\u003e \u003cp\u003eAntihypertensive treatment helps to reduce not only blood pressure (BP) values but also hypertension-induced organ damage via reduction of oxidative stress. The elevated blood pressure response to high dietary salt intake in rats has been shown to be ameliorated by some agents such as ACE inhibitors like captopril as well as the aldosterone antagonist, spironolactone, \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e implicating the Renin Angiotensin Aldosterone System (RAAS) in the response. Calcium ion Channel Blockers (CCBs) have also been used extensively to treat human hypertension either as monotherapy or in combination therapy. These compounds including the dihydropyridines (e.g. nifedipine and amlodipine) and non-dihydropyridine compounds (e.g. diltiazem and verapamil) lower blood pressure by reducing the entry of calcium ions into the heart and blood vessels However, an earlier report showed that the calcium channel antagonist, nifedipine, does not abolish the high blood pressure response to salt loading in salt diet rats.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAmlodipine which is also a member of the dihydropyridine class of calcium antagonists and is believed to have cellular action of selectively inhibiting transmembrane influx of calcium ions into vascular smooth muscle and cardiac muscle, with a greater effect on vascular smooth muscle cells than on cardiac muscle cells. In view of the reported differences in the vascular actions of different CCBs, it is possible that these agents may not be acting solely as vascular calcium ion channel blockers, in reducing blood pressure and that there may be other additional mechanisms involved. Indapamide belongs to the thiazide-like class of diuretics, and it is used as an oral antihypertensive agent where it reduces blood volume by acting on the kidneys to reduce sodium (Na\u003csup\u003e+\u003c/sup\u003e) in the distal convoluted tubule. \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Indapamide also act in the treatment of hypertension by reducing cardiac hypertrophy and thickening of arterial walls, prevents accumulation of the embryonic isoform of fibronectin in coronary vessels, scavenges free radicals, leading to stimulation of vasodilator eicosanoid formation, and interaction with renal carbonic anhydrase.\u003c/p\u003e \u003cp\u003eThe prevalence of hypertension is rising globally, and high salt intake which can cause excessive production of reactive oxygen species (ROS), overwhelming the antioxidant defense leads to increased pressure on the vascular walls and increased mechanical pressure on the membranes of red blood cells (RBCs) leading a higher erythrocyte Osmotic fragility (OF), which may be the major cause for progression to the development of cardiovascular disease (CVD) \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e and pathogenesis of hypertension.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Animals\u003c/h2\u003e \u003cp\u003eTwenty-four (24) Male Wistar rats, weighing between 185\u0026ndash;200 g were used for this study. The animals were obtained from Central Animal House, College of Medicine, University of Ibadan. They were acclimatized for 2 weeks and housed in solid bottom polypropylene cages under standard environmental conditions in a well-ventilated room under a condition of room temperature (approximately 23\u0026ndash;25 \u003csup\u003e0\u003c/sup\u003eC), relative humidity (approximately 55%) and natural environmental light (12/12- hour light/dark cycle) respectively according to the guidelines for the care and use of laboratory animals of the University of Ibadan, Nigeria. The animals were allowed access to standard grower\u0026rsquo;s feed (Top Feeds, Nigeria) and water \u003cem\u003ead libitum.\u003c/em\u003e The animals were acclimatized for two weeks and thereafter randomly divided into four groups A, B, C and D of 6 rats per group. Group A (control rats) were fed with normal feed. Group B were fed with salt supplemented diet (group B) only for 8 weeks. Group C were fed with salt supplemented feed for 8 weeks and treated with amlodipine for 4 weeks. Group D were fed with salt supplemented feed for 8 weeks and treated with indapamide for 4 weeks.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation and Administration of Drugs and Chemicals\u003c/h3\u003e\n\u003cp\u003eNorvasc Amlodipine and Indapamide were purchased from a local pharmaceutical store in Ibadan, Nigeria. 5mg/kg body weight of Amlodipine, \u003csup\u003e\u003cb\u003e11\u003c/b\u003e\u003c/sup\u003e and 1mg/kg body weight for Indapamide \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e was administered once daily for four weeks. A sodium loaded diet containing 8% NaCl was prepared by adding appropriate quantities of salt to the standard rat feed containing 0.3% NaCl (supplied by Top feeds, Ibadan).\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003eSample Collection\u003c/h3\u003e\n\u003cp\u003eOn the 28th day after drug administration, all animals were euthanized under ketamine (50mg/kg) anesthesia and blood sample were collected via cardiac puncture into Eppendorf tubes for oxidative biomarkers, \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e and into lithium heparinized tubes for hematological studies (erythrocyte osmotic fragility). The left ventricle, ascending aorta and kidney were extracted for histological examination.\u003c/p\u003e\n\u003ch3\u003eBiochemical Analysis\u003c/h3\u003e\n\u003cp\u003eMalondialdehyde (MDA) which is an end product of lipid peroxidation is determined by measuring the thiobarbituric acid reactive substances (TBARS) produced during lipid peroxidation according to the method described by Varshney and Kale. \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Reduced glutathione level (GSH) was estimated by following the protocol of Beutler \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. The level of SOD activity was determined by the method of Fridovich and Misra.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Catalase activity was determined according to the method of Sinha.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Nitric Oxide (NO) levels were measured as total nitrite plus nitrate levels using the Griess reagent as described by Moshage \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003eErythrocyte Osmotic Fragility\u003c/h3\u003e\n\u003cp\u003eErythrocyte osmotic fragility was determined based on the modified method by Oyewale \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Blood from each rat was collected into heparinized sample bottles. Serial solution of sodium chloride (pH 7.4) was prepared in the following concentrations 0.0, 0.1, 0.3, 0.5, 0.7, 0.9 g/d. 1ml of the heparinized blood was pipette into the serial solutions of sodium chloride and mixed carefully. The solution was incubated for 30 minutes at room temperature (18-25\u003csup\u003e0\u003c/sup\u003eC). The test tubes were then centrifuged at 1500 revolutions per minute (rpm) for 10 minutes using a centrifuge. The supernatant was transferred into a glass cuvette after which the concentration of the haemoglobin in the supernatant was measured calorimetrically by reading the absorbance at a wavelength of 540nm using a spectrophotometer. The percentage hemolysis was calculated using the following formula:\u003c/p\u003e \u003cp\u003ePercentage hemolysis\u0026thinsp;=\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eOptical density of test solution x 100\u003c/span\u003e\u003c/p\u003e \u003cp\u003eOptical density of water\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Tissues for Histology\u003c/h2\u003e \u003cp\u003eThe procedure was carried out according to method of Avwioro, \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of Blood Pressure and Heart Rate\u003c/h3\u003e\n\u003cp\u003eThe systolic and diastolic blood pressures were measured using a CODA non-invasive method (Kent Scientific Co., USA). The CODA tail-cuff system uses Volume Pressure recording (VPR) with specially designed differential pressure transducer to measure the blood pressure in the tail non-invasively by determining the tail volume. This is done at a temperature of 20\u003csup\u003e0\u003c/sup\u003eC after restraining the rat for at about 10\u0026ndash;15 minutes before the recording begins. VPR will actually measure six parameters simultaneously: Systolic blood pressure, Diastolic blood pressure, Mean blood pressure, Heart pulse rate, Tail blood volume and Tail blood flow.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was carried out using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Data are expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Comparison between the groups were performed by One-way and Two-way ANOVA. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThere was no significant difference in the relative weight for both the left ventricle and kidney between the control groups and the treated groups.\u003c/p\u003e\n\u003cp\u003eThe result shows a significant increase in malondialdehyde level (MDA) of the sodium fed only group when compared with the normal control group in serum (A), left ventricle (B), kidney (C) and ascending aorta (D) respectively. Significant reduction in MDA level in serum, left ventricle and kidney was observed in animals treated with sodium + amlodipine and sodium + indapamide group when compared with the sodium fed only group respectively.\u003c/p\u003e\n\u003cp\u003eResult from Figure 3 above shows that sodium fed only group showed a significant decrease in superoxide dismutase level when compared with the normal control group in serum, left ventricle, kidney and ascending aorta respectively. The group treated with sodium + amlodipine also showed a significant increase in the superoxide dismutase level only in serum, left ventricle and Ascending aorta when compared with the sodium fed only group. There was also a significant increase in the superoxide dismutase level in sodium + indapamide group when compared to the sodium fed only group in serum and ascending aorta respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResult from Figure 4 above observed that sodium fed only group showed a significant decrease in glutathione reductase level when compared with the normal control group in serum and ascending aorta respectively. More so, the groups treated with sodium + amlodipine and sodium + indapamide showed a significant decrease (in the ascending aorta) and a significant increase (in the serum) in the glutathione reductase level when compared with the sodium fed only group respectively.\u003c/p\u003e\n\u003cp\u003eResult from Figure 5, reports that sodium fed only group showed a significant decrease in catalase level in the serum and ascending aorta respectively when compared with the normal control group. The group treated with sodium + amlodipine showed a significant increase in the catalase level in the serum and ascending aorta when compared with the sodium fed only group. There was also a significant increase in the catalase level in sodium + indapamide group in both the serum and ascending aorta only when compared to the sodium fed only group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResult from Figure 6 above reports that, the sodium fed only group showed only a significant decrease in nitrite level in the left ventricle and kidney when compared with the normal control group. The groups treated with sodium + amlodipine and sodium + indapamide showed a significant decrease (in the left ventricle) and a significant increase (in the kidney) in the nitrite level when compared with the sodium fed only group respectively.\u003c/p\u003e\n\u003cp\u003eFigure 7 shows the effect of Amlodipine and Indapamide on Systolic and Diastolic blood pressures. There was a significant increase in both the Pre-treatment Systolic and diastolic blood pressure in the sodium fed only group when compared with the control group. There was also a significant decrease in the Pre-treatment Systolic and Diastolic blood pressure in the sodium fed + Amlodipine and Sodium Fed + Indapamide group when compared with the Sodium Fed only group. A significant decrease in Post treatment systolic and diastolic blood pressure was recorded in all the treated groups when compared with the Sodium Fed only group.\u003c/p\u003e\n\u003cp\u003eThere was a significant difference in the pre-treatment heart rate between the control groups and treated groups. However, a significant decrease in heart rate was recorded in the Post treatment heart rate in sodium fed + Amlodipine and sodium fed + Indapamide when compared with the sodium fed only group.\u003c/p\u003e\n\u003cp\u003eThe Sodium fed only group has significantly higher erythrocyte osmotic fragility than the Control, Sodium fed + Amlodipine and Sodium fed + Indapamide groups at 0.3, 0.5 and 0.7% of NaCl concentration, however the erythrocyte osmotic fragility in the Control, Sodium fed + Amlodipine and Sodium fed + Indapamide groups were not significantly different at this concentration. The erythrocyte osmotic fragility in the Control, Sodium fed only, Sodium fed + Amlodipine and Sodium fed + Indapamide groups were not significantly different at 0.9% of NaCl concentration.\u003c/p\u003e\n\u003cp\u003eFigure 10 show a photomicrograph of renal tissue show normal glomeruli, bowman capsule and tubules with no significant lesion seen (A). In Sodium fed rats, severe congestion of the glomeruli and several degeneration of the subcapsular tubules was observed (B). Mild renal cortical congestion was observed in Sodium fed + Amlodipine group (C). Normal glomeruli, bowman capsule and tubules with no significant lesion were observed in Sodim fed + Indapamide\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003egroup (D).\u003c/p\u003e\n\u003cp\u003eFigure 11 show a photomicrograph of cardiac tissue with normal cytoarchitecture and no significant lesion (A). Mild congestion of the coronary vessels was observed (B). Cardiac tissue shows no significant lesion (C \u0026amp; D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 12 show a photomicrograph of ascending aorta with normal cytoarchitecture and no significant lesion (A). Ascending aorta also shows several tubules with protein casts in their lumina (B). Ascending aorta shows no significant lesion (C \u0026amp; D).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study evaluated the effects of a Calcium ion Channel Blockers (CCBs)(amlodipine) and diuretics (Indapamide) on changes in oxidative stress and erythrocyte osmotic fragility of Wistar rats fed with high salt diet (8% NaCl) to induce hypertension. The results of the present study confirmed previous studies that in Wistar rats, ingestion of a high salt diet resulted in hypertension.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eExcessive production of reactive oxygen species (ROS) has been associated with a high salt intake which overwhelms the antioxidant defense and increases pressure on the vascular wall, thus increasing mechanical pressure on the membranes of red blood cells (RBCs) leading to a higher erythrocyte Osmotic fragility (OF), which may be the major cause for the progression to the development of CVD,\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e and pathogenesis of hypertension.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Result from the current study (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) shows that a high salt diet affects both the enzymatic and non-enzymatic antioxidants as well as causes membrane lipid peroxidation, thus raising malondialdehyde levels, an observation that is consistent with previous reports.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e The MDA levels in serum, kidney, ascending aorta and left ventricle in this study were significantly decreased in the sodium fed treated with amlodipine and indapamide when compared with the sodium fed only group. This decrease in MDA could be either a result of blood pressure (BP) reduction or antioxidant property of amlodipine and indapamide. These results are in agreement with various other studies that have shown MDA levels to decrease after amlodipine and indapamide administration.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOur result indicates that the superoxide dismutase (SOD) level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) in serum, kidney, ascending aorta and left ventricle at four weeks were significantly reduced in the salt-sensitive hypertensive rats (SHR) when compared with normal control group. This could be due to the fact that a high salt diet generates increased production of reactive oxygen species especially superoxide anion. The superoxide radical produced is neutralized by superoxide dismutase and the decrease in the level of this antioxidant reveals that a high salt diet leads to increased oxidative stress.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Several studies have also reported a low level of SOD in hypertensive patients. This decrease may also be associated with a decrease in NO levels, reinforcing that SOD is helpful in the release of NO, this corroborates findings from the current study. However, treatment with amlodipine and indapamide significantly increased the SOD level of these experimental rats when compared with the sodium fed only group. These findings are in agreement with various other studies that have shown SOD levels to increase after antihypertensive treatment.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eGlutathione levels in the serum, kidney, ascending aorta and left ventricle were also significantly reduced in the SHR rats when compared with the normal control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The level of reduced glutathione is a measure of the cellular redox status, hence alteration in glutathione level may affect the overall redox status of the cell. Aniya and Naito, \u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e had reported that severe oxidative stress might result in decrease in glutathione s-transferase as well as glutathione. This further confirms that there is an appreciable level of oxidative stress in the group fed with a high salt diet. The glutathione reductase levels were significantly increased in serum in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) in the sodium fed rats treated with amlodipine and indapamide groups when compared with the sodium fed only group.\u003c/p\u003e \u003cp\u003eCatalase is usually inactivated by hydrogen peroxide and superoxide radicals. Hence, reduction in residual catalase level confers increased susceptibility of the cell to undergo lipid peroxidation. This increase in lipid peroxidation is in agreement with the study carried out on hypertensive patients.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Our result further indicated that a high salt diet significantly decreased catalase activity in serum, kidney, ascending aorta and left ventricle when compared to the normal control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This could be due to some unknown mechanism in the oxidative stress process that has affected its activity. It is generally believed that hydrogen peroxide can be detoxified and removed by catalase at high concentration and glutathione peroxidase can destroys it when present at a steady state.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e The reduction in the level of this enzyme may render the kidney more susceptible to hydrogen peroxide induced oxidative stress. The catalase levels in serum and ascending aorta in this study were significantly increased in the sodium fed groups treated with amlodipine and indapamide when compared with the sodium fed only group, which clearly indicate that amlodipine and indapamide have antioxidative properties, and thus possible renoprotective properties which might be conferred through the reduction or elimination of oxidative stress in the kidney as has been shown for other antioxidants.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eResults from present study also showed that nitrite levels in the serum, kidney, ascending aorta and left ventricle were significantly reduced in the SHR rats when compared with the normal control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Several studies have reported a low level of nitrite in hypertensive patients due to an increased generation of reactive oxygen species and a down-regulation of nitric oxide synthase (NOS) subtypes.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Reactive oxygen species may act through several mechanisms to mediate vascular change in hypertension: direct actions on endothelial cells; increase quenching of nitric oxide, a vasodilator by O\u003csub\u003e2\u003c/sub\u003e-.The nitrite levels in left ventricle in this study were also further significantly decreased in the sodium fed treated with amlodipine and indapamide groups respectively when compared with the sodium fed only group. However, the nitrite level in the kidney increased in the sodium fed treated with amlodipine and indapamide groups respectively. Although the impact of different types of antihypertensive treatment on oxidative stress has been analyzed in several studies, results showed reduction in oxidative stress and an increase in antioxidant mechanisms after different antihypertensive treatments.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn the current study, the blood pressure of the rats was measured before they were placed on treatment with Amlodipine and Indapamide. The result shows a significant increase in the Pre-treatment systolic and diastolic blood pressure in the sodium fed only group when compared with normal control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), which is consistent with previous research that high sodium intake has a tendency of increasing systolic blood pressure. A significant decrease was observed in the post-treatment systolic and diastolic blood pressure of Amlodipine and Indapamide treatment groups when compared with the sodium fed only group. A significant decrease in heart rate was recorded in the post-treatment heart rate in the sodium fed treated with amlodipine group and the sodium fed treated with indapamide group when compared with sodium fed only group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRed blood cells (RBCs) being the most abundant cells in humans are the first cells to be prone to stressful stimuli, and because of their role as O\u003csub\u003e2\u003c/sub\u003e and CO\u003csub\u003e2\u003c/sub\u003e transporter, they are under constant exposure to reactive oxygen species (ROS) and oxidative stress.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e The results of the present study showed a significantly increased erythrocyte osmotic fragility (EOF) at 0.3, 0.5 and 0.7% of NaCl concentrations in the sodium fed only group compared to normal control group and sodium\u0026thinsp;+\u0026thinsp;amlodipine treated group as well as the sodium\u0026thinsp;+\u0026thinsp;indapamide treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). This result could be due to the pro-action of high salt diet on ROS level thereby resulting in lipid peroxidation of the membrane lipid bi-layers which are rich in polyunsaturated fatty acids side chains. The lipid peroxidative alteration in the structural and functional components of the erythrocyte membrane may have caused perturbations in the membrane integrity resulting in increased erythrocyte fragility recorded in the sodium fed only group in the present study. The increased EOF observed indicates the ability of the high salt diet to compromise the integrity of the erythrocyte membrane apparently from increased oxidative damage.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Our results are in agreement with previous studies that high salt diet induces oxidative stress, via the formation of free radicals and reactive oxygen species (ROS), thus increasing osmotic fragility of RBCs.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e The decrease observed in the sodium\u0026thinsp;+\u0026thinsp;amlodipine and sodium\u0026thinsp;+\u0026thinsp;indapamide treated groups could have been from the antioxidant activity of the antihypertensive therapy, which enables them to mop up the prevalent reactive oxygen species, resulting in less damage to the cell membrane of the erythrocytes. This result is in agreement with Fasanmade.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e who investigated the beneficial effects of diuretic therapy on erythrocyte osmotic fragility in hypertension during diuretic therapy and found out that erythrocyte osmotic fragility was decreased in the group that received diuretic therapy. Fujita \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e also demonstrated the ability of calcium channel blockers to offer protective effect on RBC deformability. The reduction of the EOF by antihypertensive therapy observed in the sodium\u0026thinsp;+\u0026thinsp;amlodipine and sodium\u0026thinsp;+\u0026thinsp;indapamide treated groups further confirms the role of oxidative stress in the toxic mechanism of erythrocyte damage observed in the sodium fed only group in the present study. The ability of calcium channel blockers to preserve cellular membrane integrity through its capacity to exhibit membrane modulatory effects may be responsible for this ameliorative effect.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eHistological studies reveal congestion of the interstitium of the kidney with several tubules, especially the subcapsular ones severely degenerated along with a moderate congestion of the glomeruli and several interstitial vessels in the sodium fed only group which was restored towards normal in the groups receiving the 8% NaCl diet with Amlodipine and Indapamide treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). This finding aligns with results obtained from previous studies in which male Wistar albino rats receiving 8% NaCl diet for 8 weeks displayed considerable degrees of tubular degeneration and renal tissue congestion.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e However, cardiac tissue showed mild congestion of the coronary vessels in the sodium fed only group which was restored towards normal in the groups receiving the 8% NaCl diet with Amlodipine and Indapamide treatment. This observation could be due to increased blood pressure observed in the groups fed on a high salt diet, as the elevated vascular resistance observed in hypertension has been linked to structural changes in the blood vessel (Gros et al., 2000). This finding corroborates the histoarchitectural phenotype of the coronary vessels of the rats on high-salt diet as reported by Fadahunsi and Olorunnisola.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe photomicrograph of ascending aorta showed several tubules have protein casts in their lumina in the sodium fed only group which was also restored towards normal in the groups receiving the 8% NaCl diet with Amlodipine and Indapamide treatment. The protein casts in the lumina of the aortic section may result into thickening of the wall of the aorta.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e Our findings are consistent with the reported histoarchitectural changes of the aorta of the rats on high-salt diet.\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study suggests that Amlodipine and Indapamide have an ameliorative effect on oxidative stress level of Wistar rats fed with high sodium diet. Wistar rats fed with high sodium supplemented food tend to have high blood pressure. This study also confirms the blood pressuring lowering and antioxidant effects of amlodipine, with Indapamide having a higher potency in reducing blood pressure via increasing sodium excretion.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eNa\u003csup\u003e+\u003c/sup\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;sodium\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eROS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;reactive oxygen species\u003c/p\u003e\n\u003cp\u003eSHR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;salt-sensitive hypertensive rats\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRBCs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;red blood cells\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; blood pressure\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNOS nitric oxide synthase\u003c/p\u003e\n\u003cp\u003eCCBs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;calcium channel blockers\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSOD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; superoxide dismutase SOD\u003c/p\u003e\n\u003cp\u003eCAT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;catalase\u003c/p\u003e\n\u003cp\u003eGSH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; reduced glutathione level\u003c/p\u003e\n\u003cp\u003eMDA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;malondialdehyde\u003c/p\u003e\n\u003cp\u003eEOF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;erythrocyte osmotic fragility\u003c/p\u003e\n\u003cp\u003eSF Only \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Sodium Fed\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSF + AMD \u0026nbsp; \u0026nbsp;Sodium Fed and Amlodipine\u003c/p\u003e\n\u003cp\u003eSF + IND \u0026nbsp; \u0026nbsp; \u0026nbsp; Sodium Fed and Indapamide\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNCDs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; non-communicable diseases\u003c/p\u003e\n\u003cp\u003eNOS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; nitric oxide synthase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNO \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; nitric oxide\u003c/p\u003e\n\u003cp\u003eNaCl \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; sodium chloride\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCVD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; cardiovascular diseases\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRAAS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;renin angiotensin aldosterone system\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was obtained from the Animal Care and Use Research Ethics Committee of the University of Ibadan, Nigeria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent:\u003c/strong\u003e Informed consent was obtained from all individuals included in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and agreed to the publishing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHenrietha Nwakaego Chukwuefe: Writing – original draft, Investigation, Funding acquisition, Methodology, Formal analysis, Data curation, Conceptualization.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUbong Edem David: Validation, Formal analysis, Data curation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAbodunrin Adebayo Ojetola: Writing – review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdesoji Adedipe Fasanmade: Validation, Supervision, Project administration, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMarshall IJ, Wolfe CD, McKevitt C. 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High-salt\u0026ndash;fat diet: a risk factor for elevated blood pressure associated with dyslipidemia, perturbation in cardio-renal anti-oxidant and pro-inflammatory status in Wistar rats. \u003cem\u003eJoBAZ\u003c/em\u003e 2024, 85: 62. \u003c/li\u003e\n\u003cli\u003e⁠Marelli AJ, Perloff JK, Child JS. \u003cem\u003eEt al.\u003c/em\u003e Pulmonary atresia with ventricular septal defect in adults. \u003cem\u003eCirculation\u003c/em\u003e. 1994; 89(1):243-51.\u003c/li\u003e\n\u003cli\u003eAkukwu D, Ugochukwu A, Catherine A. \u003cem\u003eet al.\u003c/em\u003e Effect of High Salt Diet and Habiscus Sabdariffa (Zobo) on the Histology of the Aorta in Wistar Rats. \u003cem\u003eInternational Research Journal of Gastroenterology and Hepatology\u003c/em\u003e 2023, 6 (1):108-20.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hypertension, Sodium supplemented diet, Amlodipine, Indapamide, antioxidants","lastPublishedDoi":"10.21203/rs.3.rs-6167252/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6167252/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe prevalence of hypertension is rising globally, and elevated blood pressure (BP) is the leading cause of death, with high salt intake as the most common cause for the development of hypertension. Amlodipine is a calcium channel blocker which is effective in the treatment of hypertension and in the prophylactic management of angina while Indapamide is a thiazide-like class of diuretics that reduces blood volume by acting on the kidneys to reduce sodium (Na\u003csup\u003e+\u003c/sup\u003e) reabsorption in the distal convoluted tubule.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTwenty-four (24) male Wistar rats weighing between 185- 200g were used for this study. The rats were randomly assigned into 4 groups (n\u0026thinsp;=\u0026thinsp;6). Group one (1) were the Normal control. Group 2: sodium supplemented diet only. Group 3 and 4: Sodium supplemented fed for 8 weeks\u0026thinsp;+\u0026thinsp;Amlodipine (5mg/kgb.w) treatment for four (4) weeks and sodium supplemented fed for 8 weeks\u0026thinsp;+\u0026thinsp;Indapamide (1mg/kgb.w) treatment for 4 weeks. After the fourth week of treatment, the animals were euthanized under ketamine (50mg/kg) anesthesia, and blood sample collected via cardiac puncture into eppendorf bottle to obtain serum for biochemical analysis and lithium heparin bottles for RBC osmotic fragility test. The left ventricle, ascending aorta and kidney were extracted for histological examination.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMalondialdehyde (MDA) level was significantly increased in Sodium fed rats when compared with control. Amlodipine and Indapamide significantly reduced MDA level in Sodium fed rats. Antioxidant enzymes were also significantly decreased in Sodium fed but was increased by Amlodipine and Indapamide respectively. The sodium fed rats exhibited significantly higher erythrocyte osmotic fragility than the control, sodium fed\u0026thinsp;+\u0026thinsp;Amlodipine and sodium fed\u0026thinsp;+\u0026thinsp;Indapamide groups at 0.3, 0.5 and 0.7% of NaCl concentration. Both systolic and diastolic blood pressures were also significantly increased in sodium fed rats when compared with control but was decreased with Amlodipine and Indapamide treatment.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eAmlodipine and Indapamide demonstrated antioxidant property and ameliorative role on Wistar rats fed with high salt diet.\u003c/p\u003e","manuscriptTitle":"Role of Calcium Channel Blockers and Diuretics in Salt -induced Oxidative Stress associated Erythrocyte Osmotic Fragility of Male Wistar rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 22:54:43","doi":"10.21203/rs.3.rs-6167252/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-05-27T05:16:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291669105148733510577860640893427924217","date":"2025-05-18T12:45:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-30T11:13:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-25T08:35:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-31T07:32:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-29T00:08:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cardiovascular Disorders","date":"2025-03-29T00:07:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b57082a4-4f90-4d3c-9d22-fe42740bec3b","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-05-05T22:54:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-05 22:54:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6167252","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6167252","identity":"rs-6167252","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}