Caveolinas y modulación del sistema del óxido nítrico en el hipotiroidismo según avanza la edad / Age-related Influence of Hypothyroidism on Caveolins and Nitric Oxide Modulation

Introducción El hipotiroidismo y la edad impactan sobre la producción de óxido nítrico (NO) cardíaco y renal. Las caveolinas, moduladores negativos de la actividad enzimática de la NO sintetasa (NOS), se afectan con ambos factores. Objetivos Evaluar la implicación de las caveolinas (cav) en la modulación de la actividad de la NOS cardíaca y renal en animales hipotiroideos adultos. Material y métodos Se utilizaron ratas macho Sprague-Dawley eutiroideas e hipotiroideas [metimazol 0,02% (v/v) en el agua de bebida durante 28 días]. Los animales fueron sacrificados para extraer el corazón y los riñones. Resultados La actividad de la NOS en la aurícula derecha disminuyó con la edad y el hipotiroidismo. La expresión de cav-1 aumentó con la edad y el hipotiroidismo. La actividad de la NOS en el ventrículo izquierdo aumentó con el avance de la edad y el hipotiroidismo. La expresión de ambas caveolinas disminuyó en los grupos adulto e hipotiroideo. En la médula renal, el hipotiroidismo disminuyó la actividad de la NOS en jóvenes y la aumentó en adultos. La expresión de cav-1 disminuyó con la edad y en jóvenes hipotiroideos. Los niveles proteicos de cav-3 disminuyeron en animales adultos hipotiroideos. Conclusiones El hipotiroidismo impacta sobre la actividad de la NOS y de sus moduladores, las caveolinas, en el sistema cardiovascular y renal. El hipotiroidismo intensifica los efectos del avance de la edad en ambos sistemas.

Introduction Hypothyroidism and age impact on cardiac and renal nitric oxide (NO) production. Caveolins, which are negative modulators of NO synthase (NOS) activity, are affected by both factors. Objectives The aim of this study was to evaluate caveolin (CAV) participation in the modulation of renal and cardiac NOS activity in adult hypothyroid animals. Methods Euthyroid and hypothyroid [methimazole 0.02% (v/v) in the drinking water during 28 days] male Sprague-Dawley rats were used. Animals were sacrificed to remove the heart and kidneys. Results Right atrial NOS activity decreased with age and hypothyroi-dism. Caveolin-1 expression increased with age and hypothyroidism. Conversely, left ventricular NOS activity increased with aging and hypothyroidism and the expression of both CAV isoforms decreased in adult and hypothyroid groups. In the renal medulla, hypothyroidism reduced NOS activity in young and raised it in adult animals and CAV-1 expression decreased with age and in hypothyroid young animals. Caveolin-3 protein levels decreased in adult hypothyroid animals. Conclusions Hypothyroidism impacts on NOS activity and on that of its modulators, caveolins, in the cardiovascular and renal systems. Hypothyroidism enhances the effects of aging in both systems.

Caveolinas y modulación del sistema del óxido nítrico en el hipotiroidismo según avanza la edad / Age-related Influence of Hypothyroidism on Caveolins and Nitric Oxide Modulation

Introducción El hipotiroidismo y la edad impactan sobre la producción de óxido nítrico (NO) cardíaco y renal. Las caveolinas, moduladores negativos de la actividad enzimática de la NO sintetasa (NOS), se afectan con ambos factores. Objetivos Evaluar la implicación de las caveolinas (cav) en la modulación de la actividad de la NOS cardíaca y renal en animales hipotiroideos adultos. Material y métodos Se utilizaron ratas macho Sprague-Dawley eutiroideas e hipotiroideas [metimazol 0,02% (v/v) en el agua de bebida durante 28 días]. Los animales fueron sacrificados para extraer el corazón y los riñones. Resultados La actividad de la NOS en la aurícula derecha disminuyó con la edad y el hipotiroidismo. La expresión de cav-1 aumentó con la edad y el hipotiroidismo. La actividad de la NOS en el ventrículo izquierdo aumentó con el avance de la edad y el hipotiroidismo. La expresión de ambas caveolinas disminuyó en los grupos adulto e hipotiroideo. En la médula renal, el hipotiroidismo disminuyó la actividad de la NOS en jóvenes y la aumentó en adultos. La expresión de cav-1 disminuyó con la edad y en jóvenes hipotiroideos. Los niveles proteicos de cav-3 disminuyeron en animales adultos hipotiroideos. Conclusiones El hipotiroidismo impacta sobre la actividad de la NOS y de sus moduladores, las caveolinas, en el sistema cardiovascular y renal. El hipotiroidismo intensifica los efectos del avance de la edad en ambos sistemas.(AU)

Introduction Hypothyroidism and age impact on cardiac and renal nitric oxide (NO) production. Caveolins, which are negative modulators of NO synthase (NOS) activity, are affected by both factors. Objectives The aim of this study was to evaluate caveolin (CAV) participation in the modulation of renal and cardiac NOS activity in adult hypothyroid animals. Methods Euthyroid and hypothyroid [methimazole 0.02% (v/v) in the drinking water during 28 days] male Sprague-Dawley rats were used. Animals were sacrificed to remove the heart and kidneys. Results Right atrial NOS activity decreased with age and hypothyroi-dism. Caveolin-1 expression increased with age and hypothyroidism. Conversely, left ventricular NOS activity increased with aging and hypothyroidism and the expression of both CAV isoforms decreased in adult and hypothyroid groups. In the renal medulla, hypothyroidism reduced NOS activity in young and raised it in adult animals and CAV-1 expression decreased with age and in hypothyroid young animals. Caveolin-3 protein levels decreased in adult hypothyroid animals. Conclusions Hypothyroidism impacts on NOS activity and on that of its modulators, caveolins, in the cardiovascular and renal systems. Hypothyroidism enhances the effects of aging in both systems.(AU)

Role of angiotensin II and oxidative stress on renal aquaporins expression in hypernatremic rats

The aim of this study was to assess whether endogenous Ang II and oxidative stress produced by acute hypertonic sodium overload may regulate the expression of aquaporin-1 (AQP-1) and aquaporin-2 (AQP-2) in the kidney. Groups of anesthetized male Sprague–Dawley rats were infused with isotonic saline solution (control) or with hypertonic saline solution (Na group, 1 M NaCl), either alone or with losartan (10 mg kg−1) or tempol (0.5 mg min−1 kg−1) during 2 h. Renal function parameters were measured. Groups of unanesthetized animals were injected intraperitoneally with hypertonic saline solution, with or without free access to water intake, Na+W, and Na−W, respectively. The expression of AQP-1, AQP-2, Ang II, eNOS, and NF-kB were evaluated in the kidney by Western blot and immunohistochemistry. AQP-2 distribution was assessed by immunofluorescence. Na group showed increased natriuresis and diuresis, and Ang II and NF-kB expression, but decreased eNOS expression. Losartan or tempol enhanced further the diuresis, and AQP-2 and eNOS expression, as well as decreased Ang II and NF-kB expression. Confocal immunofluorescence imaging revealed labeling of AQP-2 in the apical plasma membrane with less labeling in the intracellular vesicles than the apical membrane in kidney medullary collecting duct principal cells both in C and Na groups. Importantly, our data also show that losartan and tempol induces a predominantly accumulation of AQP-2 in intracellular vesicles. In unanesthetized rats, Na+W group presented increased diuresis, natriuresis, and AQP-2 expression (112 ± 25 vs 64 ± 16; *p < 0.05). Water deprivation increased plasma sodium and diuresis but decreased AQP-2 (46 ± 22 vs 112 ± 25; §p < 0.05) and eNOS expression in the kidney. This study is a novel demonstration that renal endogenous Ang II-oxidative stress, induced in vivo in hypernatremic rats by an acute sodium overload, regulates AQP-2 expression

Role of angiotensin II and oxidative stress on renal aquaporins expression in hypernatremic rats.

The aim of this study was to assess whether endogenous Ang II and oxidative stress produced by acute hypertonic sodium overload may regulate the expression of aquaporin-1 (AQP-1) and aquaporin-2 (AQP-2) in the kidney. Groups of anesthetized male Sprague-Dawley rats were infused with isotonic saline solution (control) or with hypertonic saline solution (Na group, 1 M NaCl), either alone or with losartan (10 mg kg(-1)) or tempol (0.5 mg min(-1) kg(-1)) during 2 h. Renal function parameters were measured. Groups of unanesthetized animals were injected intraperitoneally with hypertonic saline solution, with or without free access to water intake, Na+W, and Na-W, respectively. The expression of AQP-1, AQP-2, Ang II, eNOS, and NF-kB were evaluated in the kidney by Western blot and immunohistochemistry. AQP-2 distribution was assessed by immunofluorescence. Na group showed increased natriuresis and diuresis, and Ang II and NF-kB expression, but decreased eNOS expression. Losartan or tempol enhanced further the diuresis, and AQP-2 and eNOS expression, as well as decreased Ang II and NF-kB expression. Confocal immunofluorescence imaging revealed labeling of AQP-2 in the apical plasma membrane with less labeling in the intracellular vesicles than the apical membrane in kidney medullary collecting duct principal cells both in C and Na groups. Importantly, our data also show that losartan and tempol induces a predominantly accumulation of AQP-2 in intracellular vesicles. In unanesthetized rats, Na+W group presented increased diuresis, natriuresis, and AQP-2 expression (112 ± 25 vs 64 ± 16; *p < 0.05). Water deprivation increased plasma sodium and diuresis but decreased AQP-2 (46 ± 22 vs 112 ± 25; §p < 0.05) and eNOS expression in the kidney. This study is a novel demonstration that renal endogenous Ang II-oxidative stress, induced in vivo in hypernatremic rats by an acute sodium overload, regulates AQP-2 expression.

Nitric oxide and AQP2 in hypothyroid rats: a link between aging and water homeostasis.

OBJECTIVE: Hypothyroid state and aging are associated with impairment in water reabsorption and changes in aquaporin water channel type 2 (AQP2). Nitric oxide (NO) is involved in AQP2 trafficking to the apical plasma membrane in medullary collecting duct cells. The purpose of this study was to investigate whether aging and hypothyroidism alter renal function, and whether medullary NO and AQP2 are implicated in maintaining water homeostasis. MATERIALS/METHODS: Sprague-Dawley rats aged 2 and 18months old were treated with 0.02% methimazole (w/v) during 28days. Renal function was examined and NO synthase (NOS) activity ([(14)C (U)]-L-arginine to [(14)C (U)]-L-citrulline assays), NOS, caveolin-1 and -3 and AQP2 protein levels were determined in medullary tissue (Western blot). Plasma membrane fraction and intracellular vesicle fraction of AQP2 were evaluated by Western blot and immunohistochemistry. RESULTS: A divergent response was observed in hypothyroid rats: while young rats exhibited polyuria with decreased medullary NOS activity, adult rats exhibited a decrease in urine output with increased NOS activity. AQP2 was increased with hypothyroidism, but while young rats exhibited increased AQP2 in plasma membrane, adult rats did so in the cytosolic site. CONCLUSIONS: Hypothyroidism contributes in a differential way to aging-induced changes in renal function, and medullary NO and AQP2 would be implicated in maintaining water homeostasis.

Contribution of caveolin-1 to ventricular nitric oxide in age-related adaptation to hypovolemic state.

Our previous results have shown that hypovolemic state induced by acute hemorrhage in young anesthetized rats triggers heterogeneous and dynamic nitric oxide synthase (NOS) activation, modulating the cardiovascular response. Involvement of the nitric oxide pathway is both isoform-specific and time-dependent. The aim of the present study was to investigate changes in activity and protein levels of the different NOS forms, changes in the abundance of caveolin-1 during hypovolemic state and caveolin-1/eNOS association using young and middle-aged rats. Therefore, we studied (i) changes in NOS activity and protein levels and (ii) caveolin-1 abundance, as well as its association with endothelial NOS (eNOS) in ventricles from young and middle-aged rats during hypovolemic state. We used 2-month (young) and 12-month (middle-aged) old male Sprague-Dawley rats. Animals were divided into two groups (n=14/group): (a) sham; (b) hemorrhaged animals (20% blood loss). With advancing age, we observed an increase in ventricle NOS activity accompanied by a decrease in eNOS and caveolin-1 protein levels, but increased inducible NOS (iNOS). We also observed that aging is associated with caveolin-1 dissociation from eNOS. Myocardia from young and middle-aged rats subjected to hemorrhage-induced hypovolemia exhibited an increase in NOS activity and protein levels with a reduction in caveolin-1 abundance, accompanied by a greater dissociation between eNOS and its regulatory protein. Further, an increase in iNOS protein levels after blood loss was observed only in middle-aged rats. Our evidence suggests that aging and acute hemorrhage contribute to the development of upregulation in NOS activity. Our findings demonstrate that specific expression patterns of ventricular NOS isoforms, alterations in the amount of caveolin-1 and caveolin-1/eNOS interaction are involved in aged-related adjustment to hypovolemic state.

Hypothyroidism: age-related influence on cardiovascular nitric oxide system in rats.

This study investigates whether changes in nitric oxide (NO) production participate in the cardiovascular manifestations of hypothyroidism and whether these changes are age-related. Sprague-Dawley rats aged 2 and 18 months old were treated with 0.02% methimazole (wt/vol) during 28 days. Left ventricular function was evaluated by echocardiography. Measurements of arterial blood pressure, heart rate, nitric oxide synthase (NOS) activity and NOS/caveolin-1 and -3 protein levels were performed. Hypothyroidism enhanced the age-related changes in heart function. Hypothyroid state decreased atrial NOS activity in both young and adult rats, associated with a reduction in protein levels of the three NOS isoforms in young animals and increased caveolin (cav) 1 expression in adult rats. Ventricle and aorta NOS activity increased in young and adult hypothyroid animals. In ventricle, changes in NOS activity were accompanied by an increase in inducible NOS isoform in young rats and by an increase in caveolins expression in adult rats. Greater aorta NOS activity level in young and in adult Hypo rats would derive from the inducible and the endothelial NOS isoform, respectively. Thyroid hormones would be one of the factors involved in the modulation of cardiovascular NO production and caveolin-1 and -3 tissue-specific abundance, regardless of age. Hypothyroidism appears to contribute in a differential way to aging-induced changes in the myocardium and aorta tissues. Low thyroid hormones levels would enhance the aging effect on the heart. Age-related changes in NO production participate in the cardiovascular manifestations of hypothyroidism.

Caveolinas cardíacas y modulación de la óxido nítrico sintetasa durante el estado hipovolémico según avanza la edad / Age-Related Changes of Cardiac Caveolins and Nitric Oxide Synthase Modulation during Hypovolemic State / Age-Related Changes of Cardiac Caveolins and Nitric Oxide Synthase Modulation during Hypovolemic State

Introducción El óxido nítrico (NO) es uno de los factores que se estimulan durante el estado hipovolémico. Las caveolinas afectarían la producción de NO en el corazón según avanza la edad. Objetivo Investigar la interacción entre la caveolina-1 (cav-1) y la óxido nítrico sintetasa endotelial (eNOS) en ventrículos de ratas jóvenes y adultas sometidas a hemorragia aguda. Material y métodos Se utilizaron ratas Sprague-Dawley machos jóvenes (2 meses de edad) y adultas (12 meses de edad). Se conformaron dos grupos experimentales de cada grupo etario (jóvenes y adultas): Control y Hemo (sangrado: 20% de la volemia). Los animales se sacrificaron para la extracción del ventrículo izquierdo. Resultados Luego del sangrado, la actividad de la NOS aumentó el 21% y el 45% en ratas jóvenes y el 32% y el 56% en adultas. El Western blot reveló que los niveles de eNOS fueron menores (31%) en ratas adultas. La hemorragia ocasionó un aumento del 147% de la eNOS a los 60 min en animales jóvenes y del 66% en adultos, atenuándose a los 120 min. La colocalización mostró un patrón difuso de localización de la eNOS asociado con cav-1 en animales del grupo Control jóvenes. En el grupo Hemo, a los 60 min se observó un patrón de disociación, el cual fue parcialmente restablecido a los 120 min. En los animales adultos del grupo Control se observó un patrón de localización de la eNOS y cav-1 con una disociación mayor que en las ratas jóvenes. Esta característica se observó también luego de los 60 y los 120 min. Conclusión El aumento de la producción de NO ante un estado hipovolémico se ve condicionado por la presencia de cav-1, la cual cumple un papel preponderante en el proceso del envejecimiento.

Background Nitric Oxide (NO) is one of the factors activated by hypovolemic state. Caveolins might affect the production of NO with increasing age. Objective To investigate the interaction between caveolin-1 (cav-1) and endothelial nitric oxide synthase (eNOS) in the ventricles of young and adult rats subjected to acute bleeding. Material and Methods We used young (2 months old) and adult (12 months old) male Sprague-Dawley rats. The animals from each age group (young and adult) were divided into two experimental groups: Control: control group, and Hem: bleeding group (20% of blood volume). The animals were sacrificed and the left ventricle was resected. Results After bleeding, NOS activity increased by 21% and 45% in young rats and by 32% and 56% in adult rats. Western blot analysis revealed that NOS levels were lower (31%) in adult rats. Sixty minutes after bleeding, eNOS activity increased by 147% in young animals and by 66% in adults, with lower increase at 120 minutes. Colocalization showed a diffuse pattern of eNOS location associated with cav-1 in group Control of young animals. A dissociation pattern was observed in group Hem at 60 min which was partially restored at 120 min. The dissociation pattern of eNOS and cav-1 in group Control adult animals was greater compared to that of young rats. This characteristic was also seen after 60 min and 120 min. Conclusion The increased production of NO during hypovolemic state depends on the presence of cav-1 which plays a key role in the aging process.