SUPPLEMENTATION OF ALPHA-TOCOPHEROL IS ABLE TO MODULATE HEART AND KIDNEY HISTOPATHOLOGICAL FEATURES OF SHRSP RATS

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1 CURRENT TOPICS IN NUTRACEUTICAL RESEARCH Vol. 12, No. 1/2, pp , 2014 ISSN print, Copyright 2014 by New Century Health Publishers, LLC All rights of reproduction in any form reserved SUPPLEMENTATION OF ALPHA-TOCOPHEROL IS ABLE TO MODULATE HEART AND KIDNEY HISTOPATHOLOGICAL FEATURES OF SHRSP RATS 1 Marcela R. M. Guimarães, 1 Leonardo B. Murad, 2 Aline Paganelli, 2 Carlos Alberto Basílio de Oliveira and 1 Lucia M. A. Vianna 1 Laboratory of Nutritional Investigation and Degenerative-Chronic Diseases, (LINDCD) Federal University of Rio de Janeiro State UNIRIO. Rio de Janeiro, RJ, Brazil; and 2 Pathology Laboratory - University Hospital Gaffreé and Guinle (HUGG) UNIRIO. Rio de Janeiro, RJ, Brazil. [Received November 6, 2013; Accepted: March 6, 2014] [Communicated by Arrigo F.G. Cicero, MD, PhD] ABSTRACT: We investigate whether alpha-tocopherol treatment is able to modulate the affected heart and kidney structures of severely hypertensive animals. SHRSP rats subdivided into two groups receiving either 120IU of alphatocopherol acetate or control. Systolic blood pressure was measure twice a week. Afterwards, the rats were sacrificed and the tissues were analyzed by histopathology. Oxidative stress was performed by measuring the levels of plasma malondialdehyde. There was a significant hypotensive effect ( ± 2.04 mmhg to ± 0.05 mmhg, p<0.05) and sharp decrease of malondialdehyde levels (4.55 ±0.12 nmol/dl vs ± 0.21 nmol/dl, p<0.05) in animals treated with alpha-tocopherol compared to controls. According to the heart histopathology, the average number of muscle fibers in the treated group was lower than control group (41.2 ± 4.08 cells vs ± 3.63 cells, p<0.05). Regarding renal histopathology it was observed that there was a significant decrease of hyalinization of the glomeruli and renal vessel in the treated group vs. control group (34.9 ± 4.92 vs ± 7.44, p<0.05) and (24.25±4.87 vs ±9.84, p<0.05). This study suggests that alpha-tocopherol may arouse interest in the treatment of vascular diseases. KEY WORDS: Alpha-tocopherol, Antioxidant, Heart histopathology, Renal histopathology, SHRSP rats Corresponding Author: Lucia M. A. Vianna Federal University of Rio de Janeiro State (UNIRIO). Laboratory of Nutritional Investigation and Degenerative-Chronic Diseases, Xavier Sigaud Street, 290 Urca. Zip Code: Rio de Janeiro City, Rio de Janeiro State, Brazil. lindcd@ig.com.br INTRODUCTION Hypertension is directly associated with morbidity and mortality from stroke, myocardial infarction and renal failure (Leong et al., 2008). The antihypertensive treatment is made not only in order to decrease arterial blood pressure, but also to attenuate and prevent myocardial changes that occur in hypertension, characterized by hypertrophy of cardiomyocytes, interstitial and perivascular fibrosis, as well as the decrease in the ratio intramyocardial artery - myocytes (Amann et al., 2002). It is also important to note that there is a peculiar relationship between the. Kidney and arterial blood pressure while renal dysfunction causes an increase in arterial blood pressure and this accelerates the loss of renal function (Adamcza et al., 2002). In this context, oxidative stress seems to be closely involved in the development of several pathologies, which include among others: hypertension, atherosclerosis, cardiac hypertrophy and stroke. In large measure, this fact is a consequence of an excessive production of reactive oxygen substances in face of a decrease of antioxidant capacity (Touyz, 2003; Tain and Baylis, 2006; Vaziri and Rodriguez-Iturbe, 2006). Thereafter, vitamin E has been studied as one of the most abundant fat-soluble antioxidants found in plasma and cells of higher mammals. This vitamin exists in the forms of tocopherols and tocotrienols being alpha-tocopherol considered the most biologically active (Rigotti, 2007). Alphatocopherol is associated with the prevention of various diseases because of its action on cell membranes protection from lipid peroxidation by scavenging free radicals (Herrera and Barbas, 2001; Dutta and Dutta, 2003). However, in spite of all the benefits that alpha-tocopherol might have its role on heart and kidney histology in not

2 20 Alpha-tocopherol and vascular diseases completely understood. Therefore the main reason for this study was to investigate whether alpha-tocopherol treatment is able to modulate the affected heart and kidney structures of severe hypertensive animals. MATERIAL AND METHODS Animals. Ten male rats SHRSP (spontaneously hypertensive stroke-prone rats), 8 weeks old, weighing g, were maintained in metabolic cages in a bioterium of the State Federal University of Rio de Janeiro (UNIRIO) under controlled environmental conditions: temperature (21±2 C), light cycle kept dark for 12 hours with artificial light (7:00am - 7:00pm), humidity (60±10%) and air exhaustion cycle (15 min /h). All animals received foods pellets rat chow (Nuvilab from Nuvital Co) and water ad libitum. The procedures followed the rules of the conventional guide for animal experimentation (Publication No.85-23, NIH, revised 1996). We also used the experimental protocol approved by the ethical committee for animal experimentation of the Federal University of Rio de Janeiro State, and tests were performed at the Laboratory of Nutritional Investigation and Degenerative-Chronic Diseases. Supplementation. The animals were kept in the baseline during ten days after being divided into two groups of five animals each: Group AT-treated with 120IU of alphatocopherol acetate (Sigma T-3376, St. Louis, MO) and control group receiving 0.1 ml of coconut oil, vehicle solution of alpha-tocopherol. Supplementation occurred by orogastric gavage using a polyethylene catheter PE 190 during a period of 4 weeks. Physiological parameters and blood pressure. The rats were evaluated daily for the food and water intake, urine output, body weight and physical aspects (skin, mucosa and hair), posture and behavior. Systolic blood pressure was measured by plethysmography twice a week. performed using colorimetry to calculate the concentration of MDA from the absorption of light at the 532 nm wavelength. The values were presented in nmol/ml. Histopathologic analysis. All removed material was stored in formaldehyde solution 10% and sent for histopathological analysis, performed at the Laboratory of Pathology, University Hospital Gaffrée Guinle, Federal University of Rio de Janeiro State. With the material (heart and kidneys), the slides were prepared for microscopic analysis, being carried cut 5 microns thick, the Gung RM 2025 microtome (Leica, Nussloch, Germany). The specimens were stained with hematoxylineosin. Morphometric analysis was performed using an optical microscope (Zeiss Axioplan, Zeiss, Germany) under magnification of 10X, 40X and 60X. Cardiac fibers were observed in cross-section samples from 50 fields per slide at high magnification/400x. In the kidneys, the number of hyalinization of glomeruli and vessels were observed in cross section samples from 50 fields per slide at high magnification/400x. Statistical analysis. The data were presented as mean ± standard derivation, and applied the Student s t test for comparison between treated and control groups. P<0.05 was considered statistically significant. RESULTS In agreement with previous findings from our laboratory (Guimarães et al., 2009) the oral supplementation with 120 IU of alpha-tocopherol did not change the general biological parameters (table 1); however there was a significant hypotensive effect since systolic blood pressure decreased from ± 2.04 mmhg to ± 0.05 mmhg (p<0.05) (Figure 1) and by a significant decrease of malondialdehyde levels: 4.55 ±0.12 nmol/dl vs ± 0.21 nmol/dl (Figure 2), as observed in the study cited above. Sacrifice and collection of material. After the experiment period, rats of both groups were in deep coma induced by administration of barbiturates (thiopental sodium) intraperitoneally at doses above 25 mg/kg. Thereafter, blood was collected via cardiac puncture. Blood was collected from each rat and placed in a tube, and the tubes were then centrifuged at 2000 rpm for 10 minutes to obtain the serum. This procedure also allowed the removal of the heart and kidneys to then carry out the histopathological analysis. Oxidative stress marker. The evaluation of oxidative stress was performed by measuring the levels of plasma malondialdehyde (MDA), which is a marker of lipid peroxidation. For this assay, we used thiobarbituric acid and Micronal spectrophotometer model B-442 (São Paulo, SP, Brazil). The analysis was TABLE I. General Physiological parameters Physiological parameters Alpha -Tocopherol Control Group Group Body weight (g) ± ±17.1 Diuresis (ml) 2.94± ±1.41 Water intake (ml) 29.54± ±1.01 Food intake (g) 16.86± ±3.12 According to the heart histopathology, it can be seen that the average number of muscle fibers in the treated group is lower (41.2 ± 4.08 cells) than the average in the control group (56.8 ± 3.63 cells) p<0.05 (Figure3), suggesting a protection in cardiac tissue. Regarding the renal histopathology it was observed that there was a significant decrease of hyalinization of the glomeruli of the treated group compared to the control

3 Alpha-tocopherol and vascular diseases 21 group: 34.9 ± 4.92 vs ± 7.44, p<0.05 (Figure 4), and a decrease of hyalinization of the renal vessels, ± 4.87 vs ±9.84, p<0.05 (Figure 5). The figures 6 illustrates the results of histopathological analyses. FIGURE 4. Number of hyalinized glomeruli (mean ± standard derivation) between groups. *p< considered significative difference. DISCUSSION This study confirms that SHRSP rats are responsive to alphatocopherol supplementation and the mechanism most likely to explain the hypotensive tocopherol induced effect seems to be associated to the antioxidant property of this vitamin. The reduction of oxidative stress certainly explains the hypotensive FIGURE 1. The values represent the mean and standard derivation of systolic blood pressure of control (n = 5) and treated (n = 5) over the weeks of the experiment. *p<0.05 considered significative difference. FIGURE 5. Number of renal vessels hyalinized groups. *p< considered significative difference. FIGURE 2. Levels of plasma malondialdeyde (mean ± standard derivation) between groups. *p<0.05 considered significative difference. FIGURE 3. Counting of cardiomyocytes per field (mean ± standard derivation) between groups. *p<0.05 considered significative difference. action of alpha-tocopherol, in which case all the mechanisms associated with the endothelial injury and an imbalance between the secretion of vasoconstrictor and vasorelaxant are prevented. Thus, Noguchi et al (2001) previously reported a hypotensive effect accompanied by lower urinary excretion of 8-hydroxy- 2-deoxyguanosine, a marker of oxidative stress, in SHRSP rats treated with this vitamin. Other authors observed a reduction of NADPH oxidase activity and an increased activity of superoxide dismutase under tocopherol treatment (Chen et al., 2002). Once again, tocopherol treatment provoked a sharply decreased of blood malondialdehyde levels, a potent marker of lipid peroxidation. Additionally, alpha-tocopherol treatment demonstrated an important protective action against heart and kidney damage usually presented by SHRSP. Both cardiac and kidney histology revealed a significant decrease in the number of cardiomyocytes, hyalinized renal vessels and glomeruli in the treated group. Similarly, other authors have found beneficial effects of alpha-tocopherol supplementation in renal tissue damage in different animal models (Rhoden et al., 2001; Tian et al., 2005; Kutlubay et al., 2007). Previously Otani et al (1999) also demonstrated that supplemental dietary vitamin E suppresses mesangial cell proliferation and glomerular sclerosis in models of glomerular disease in rats. The authors suggested that mesangial cells

4 22 Alpha-tocopherol and vascular diseases proliferation was suppressed via the inhibition of intracellular transduction, for example, that regulating protein kinase C. Moreover, it has been found not to be the consequence of a decreased protein expression (Tasinato et al., 1995). the authors, this result set might be due to their scavenging free radicals, enhancing antioxidant system and restoring Ca2++ levels. In fact, oxidative stress depresses the Ca2++ transport and results in the development of intracellular Ca2++ overload FIGURE 6. A comparative histopathological analysis of cardiomyocytes and renal tissue in the control group and in treated group. A: Histopathological analysis of cardiomyocytes in treated group. The cardiomyocytes are without abnormality. B: Histopathological analysis of cardiomyocytes in control group. The cardiomyocytes are with hypertrophy. C: Renal histopathological, referring to the glomeruli in treated group, with preserved glomeruli. D: Renal histopathological, referring to the glomeruli in control group, with sclerotic glomeruli. E: Renal histopathological, referring to the vessels wall in treated group. Blood vessels are with preserved wall. F: Renal histopathological, referring to the vessels wall in control group. Blood vessels are with thickened wall. Ricciarelli et al (1998) observed that alpha-tocopherol specifically inactivates cellular protein kinase C alpha by changing its phosphorylation state. According to these authors, the inhibition of protein kinase C seems to be linked to an alpha-tocopherol-induced decrease of its permissive phosphorylation. This even seems to be linked to the activation by alpha-tocopherol of protein phosphatase 2A. It is important to recognize that kidney mesangial cells and vascular smooth muscle cells are closely related in terms of origin, microscopic anatomy, histochemistry, and contractility and this relationship suggests a similarity between kidney glomerular sclerosis and atherosclerosis (Mune et al., 2002) which emphasizes the importance of these findings. The observed cardioprotective effects of alpha-tocopherol were also seen by Punithavathi and Prince (2010). According to and ventricular dysfunction (Tappia et al., 2001). The limitation of this study can be related to the fact that we used an animal model. In these types of studies, the sample size is always reduced and this may be considered a limitation. However, all stages of the work were controlled by the researchers and there were no external factors to harm the progress of the experiment. Indeed, this study goes toward a direction that alphatocopherol as an antioxidant nutrient, can arouse interest in future use as it is a contributing factor in the treatment of vascular diseases. ACKNOWLEDGEMENTS This work received support from UNIRIO, CNPq and CAPES.

5 Alpha-tocopherol and vascular diseases 23 CONFLICT OF INTEREST There is no conflict of interest. REFERENCES Adamcza M., Zeier M., Dikow R. and Ritz E. (2002). Kidney and hypertension. Kidney International Supplements 85: Amann K., Tornig J., Buzello M., Kuhlmann A., Gross M.L., Adamcza M., Buzelo M. and Ritz E. (2002). Effect of antioxidant therapy with dl-α-tocopherol on cardiovascular structure in experimental renal failure. Kidney International 62: Chen J., He J., Hamm L., Batuman V. and Whelton P.K. (2002). Serum antioxidant vitamins and blood pressure in the United States population. Hypertension 40: Costa V.A. and Vianna L.M. (2005). Effects of alphatocopherol supplementation on blood pressure and lipidic profile in streptozotocin-induced diabetes mellitus in spontaneously hypertension rats. Clinica Chimica Acta 351: Costa V.A., Vianna L.M., Aguila M. and Mandarim-De- Lacerda C.A. (2005). Alpha-tocopherol supplementation favorable effects on blood pressure, blood viscosity and cardiac remodeling of spontaneously hypertensive rats. The Journal of Nutritional Biochemistry 16: Dutta A. and Dutta S.K. (2003). Vitamin E and its role in the prevention of atherosclerosis and carcinogenesis: a review. Journal of the American College of Nutrition 22: Guimarães M.R.M., Murad L.B., Castro R.B. and Vianna L.M. (2009) Oral treatment with alpha-tocopherol reduce systolic blood pressure, oxidative stress and modulate lipid profile on spontaneously hypertensive rats stroke prone. Current Topic in Nutraceutical Research 7: Herrera E. and Barbas C. (2001). Vitamin E: action, metabolism and perspectives. Journal of Physiology and Biochemistry 57: Kutlubay Y.R., Oguz E.O., Guven C., Can B., Sinik Z. and Tunkay O.L. (2007). Histological and ultrastructural evidence for protective effects on aluminium-induced kidney damage by intraperitoneal administration of alpha-tocopherol. International Journal of Toxicology 26: Leong X.F., Aishah A., Aini U.N., Das S. and Jaarin, K. (2008). Heated palm oil causes rise in blood pressure and cardiac changes in heart muscle in experimental rats. Archives of Medical Research 39: Mune M., Otani H. and Yukawa S. (2002). Effects of antioxidants on kidney disease. Mechanism of Aging and Development 123: Noguchi T., Ikeda K., Sasaki Y., Yamamoto J., Seki J., Yamagata K., Nara Y., Hara H., Kakuta H. and Yamori Y. (2001). Effects of vitamin E and sesamin on hypertension and cerebral thrombogenesis in stroke-prone spontaneously hypertensive rats. Hypertension Research 24: Otani H., Mune M., Yukawa S., Smith D., Meydani M. and Blumberg J (1999). Vitamin E treatment of experimental glomerular disease in rats. Kidney International 56: Punithavathi V.R. and Prince P.S.M. (2010). The cardioprotective effects of the combination of quercetin and α tocopherol on isoproterenol-induced myocardial infected rats. Journal of Biochemical and Molecular Toxicology 25: Ricciarelli R., Tasinato A., Clement S., Ozer N.K., Boscoboinik D. and Azzi A. (1998). α-tocopherol specifically inactivates cellular protein kinase C α by changing its phosphorylation state. Biochemical Journal 334: Rigotti A. (2007). Absorption, transport and tissue delivery of vitamin E. Molecular Aspects of Medicine 28: Rhoden E.L., Pereira-Lima L., Teloken C., Lucas M.L., Bello-Klein A. and Rhoden C.R. (2001). Beneficial effects of α-tocopherol in renal ischemia-reperfusion in rats. Japanese Journal of Pharmacology 87: Santos R.S., Costa V.A. and Vianna L.M. (2007). Cholecalciferol treatment changes urinary sodium-potassium ratio and plasma aldosterone of spontaneously hypertensive rats. Clinica Chimica Acta 376: Tain Y.L. and Baylis C. (2006). Dissecting the causes of oxidative stress an in vivo model of hypertension. Hypertension 48: Tappia P.S., Hata T., Hozaima L., Sanndhh M.S., Panagia V. and Dhallia N.S. (2001). Role of oxidative stress in cathecholamines-induced changes in cardiac sarcolemmal Ca2++ transport. Archives of Biochemistry and Biophysics 387: Tasinato A., Boscoboinik D., Bartoli G.M., Maroni P. and Azzi A. (1995). d-α Tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties. Proceedings of the National Academy of Sciences 92: Tian N., Thrasher K.D., Gundy P.D., Hughson M.D. and Manning R.D. (2005). Antioxidant treatment prevents renal

6 24 Alpha-tocopherol and vascular diseases damage and dysfunction and reduces arterial pressure in saltsensitive hypertension. Hypertension 45: Touyz R.M. (2003). Reactive oxygen species in vascular biology: role in arterial hypertension. Expert Review of Cardiovascular Therapy 1: Vaziri N.D. and Rodriguez-Iturbe B. (2006). Mechanisms of disease: oxidative stress and inflammation in the pathogenesis of hypertension. Nature Clinical Practice Nephrology 2: