RESUMEN
OBJECTIVE: The present study examines whether a long-term high salt diet causes hypertension and renal injury in normal subjects [Sprague-Dawley (SD) rats] and alters renal cytokine-related gene expression profiles. METHODS: Four 10 week old male SD rats received a high salt diet (HS, 8%) and the other 4 SD rats received a normal salt diet (NS, 0.5%) for 8 weeks. Mean arterial pressure (MAP) and renal damages such as albuminuria and histological renal injury were determined. The relative mRNA levels of 514 cytokine-related genes (normalized by beta-actin) in rat kidneys following NS or HS were determined quantitatively through analysis of 4 sets of gene expression profiles using the mouse cDNA membrane microarrays. RESULTS: We demonstrated that 8 weeks of HS diet increased MAP [(140.0+/-5.3) vs (112.0+/-2.2) mmHg; 1 mmHg=0.133 kPa, P<0.01], albuminuria [(41.4+/-3.2) vs (20.1+/-4.5) mg/d; P<0.01], and caused histological renal injury in SD rats, compared to NS group. Of the 514 genes in the array, there were 27 (5.25%) genes with significantly different expression in the kidney of SD rats with HS compared to those of SD rats with NS. Functional clustering analysis indicated the following functional pathways related to high salt diet-induced hypertension: (1) pro-inflammatory response ( upward arrowIL-17, CCL28; downward arrow NFkappabib); (2) endothelial dysfunction ( downward arrowVEGF-A, VEGF-B, endoglin); (3) pro-matrix formation ( upward arrowosteopontin, IGFBP-5; downward arrow IFN-gamma); and (4) attenuated cell survival and differentiation ( downward arrowCNTF, IGF-II R, ephrin-B1). Northern blot confirmed that 8 weeks of HS diet significantly decreased renal expression of VEGF mRNA, compared to NS group (P<0.01). ELISA showed that HS diet significantly decreased renal protein levels of VEGF and CCL28. CONCLUSION: These findings support the hypothesis that hypertension can be induced in normal rats by a long-term high salt diet, which is associated with increased renal injury and marked changes in renal cytokine gene expression profiles that are closely related to the pro-inflammatory response, pro-matrix formation, endothelial dysfunction, and attenuated cell survival and differentiation.
Asunto(s)
Citocinas/genética , Perfilación de la Expresión Génica , Hipertensión/etiología , Riñón/metabolismo , Sodio en la Dieta/efectos adversos , Albuminuria/etiología , Albuminuria/metabolismo , Animales , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Citocinas/metabolismo , Riñón/patología , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sodio en la Dieta/administración & dosificación , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Clinical evidence links the inhibition of VEGF to hypertension. However, the mechanisms by which VEGF affects the pathogenesis of hypertension remain in question. We determined 1) whether administration of VEGF receptor inhibitor SU5416 enhances dietary salt-induced hypertension in Sprague-Dawley (SD) rats, and 2) whether VEGF or SU5416 directly affects proliferation of cultured human renal proximal tubular epithelial cells (HRPTEC) and endothelial nitric oxide synthase (eNOS) expression in cultured human glomerular microvessel endothelial cells (HGMEC). Ten 10-wk-old male SD rats received a high sodium diet (HS; 8%) and the other 10 SD rats received a normal sodium diet (NS; 0.5%) for 4 wks. After 2 wks of the dietary program, five rats were administered with SU5416 at 10 mg x kg(-1) x day(-1) ip or DMSO (vehicle) for 14 days in HS and NS groups. Mean arterial pressure was significantly higher in rats treated with SU5416, as opposed to those treated with DMSO and fed with HS for 4 wk (157.6 +/- 3.9 vs. 125.9 +/- 4.3 mmHg, P < 0.01). Increased proteinuria and albuminuria were associated with marked renal histological abnormalities in HS group with SU5416 administration, compared with those in the vehicle HS group. 3H-thymidine incorporation assay showed that SU5416 blocked the actions of both exogenous and endogenous VEGF on the proliferation of HRPTEC. VEGF (10 ng/ml) significantly increased eNOS protein levels by 29% in cultured HGMEC, but its action was completely abolished by SU5416. These results suggest that VEGF receptor inhibition enhances dietary salt-induced hypertension and kidney injury, possibly by direct damage on renal cells and decreasing NO production by eNOS.
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Inhibidores de la Angiogénesis/farmacología , Presión Sanguínea/efectos de los fármacos , Hipertensión/etiología , Indoles/farmacología , Riñón/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/farmacología , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Albuminuria/etiología , Albuminuria/metabolismo , Albuminuria/fisiopatología , Inhibidores de la Angiogénesis/administración & dosificación , Animales , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Hipertensión/metabolismo , Hipertensión/fisiopatología , Indoles/administración & dosificación , Inyecciones Intraperitoneales , Riñón/metabolismo , Riñón/patología , Riñón/fisiopatología , Glomérulos Renales/irrigación sanguínea , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Masculino , Natriuresis/efectos de los fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Pirroles/administración & dosificación , Ratas , Ratas Sprague-Dawley , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Cloruro de Sodio Dietético , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We seek to determine: 1) whether a long-term high salt diet induces hypertension and renal injury in Sprague-Dawley (SD) rats and 2) whether the high salt diet-induced hypertension and renal injury are associated with decreased renal VEGF expression. Twelve 10-wk-old male SD rats received a high salt diet (HS, 8%) and twelve SD rats received a normal salt diet (NS, 0.5 %) for 8 weeks. Using a tail cuff, weekly monitoring showed that blood pressure increased significantly after 6, 7, & 8 wks in HS group, compared to NS group (P<0.01). At 4 wks and 8 wks of diet, mean arterial pressure (MAP) was determined in conscious rats by continuous monitoring through a catheter placed in the carotid artery. MAP was not significantly different between HS and NS group in 4 wks, but was significantly higher in HS than NS group (140+/-5.3 vs.112+/-2.2 mmHg; P<0.01) in 8 wks. Increased proteinuria and albuminuria were associated with marked renal histological abnormalities in HS group, compared to those in NS group. Northern blot and ELISA demonstrated that 8 wks of HS diet significantly decreased renal expression of VEGF mRNA and protein, compared to NS group (P<0.01). In 8 wks, total urinary excretion of sFlt-1 was significantly higher in HS than NS group (9.28+/-1.05 vs. 2.05+/-0.55 ng/day; P<0.01) whereas the plasma levels of sFlt-1 remained stable. These results suggest that a long-term HS diet induces renal injury and hypertension, which are associated with decreased renal VEGF expression in normotensive rodent animals.
RESUMEN
The mechanisms of alcohol-induced cancer in humans are unclear. We used the immunocompetent mice implanted with B16F10 cells to evaluate the effects of physiologically relevant EtOH intake on tumor growth and angiogenesis of melanoma. Six-wk-old male mice (C57BL/6J) were given 1% EtOH in drinking water for 12-hrs during the night which was then replaced with regular water during the remaining 12-hrs each day for 4 wks (n = 10). The control mice received regular drinking water only. In the second week, all mice were inoculated subcutaneously on the right proximal dorsal with 5 x 10(5) B16F10 cells. In the end, the tumors were isolated for measuring tumor size, average microvascular density (AMVD) using CD31 immunohistochemistry, and the expression of VEGF and its receptor (Flt-1) using Northern blot, ELISA, and immunohistochemistry. EtOH intake caused a 2.16-fold increase in tumor weight over the control (4.81 +/- 0.39 vs. 2.23 +/- 0.48 g; n = 10; p = 0.003), a 2.02-fold increase in AMVD (60.63 +/- 5.56 vs. 30.01 +/- 7.41/mm(2); p = 0.0014), and a significant increase in VEGF mRNA and protein expression plus Flt-1 protein levels in melanoma compared to the control group (p < 0.01). These results suggest that progression of melanoma growth and angiogenesis may be mediated by upregulation of VEGF and Flt-1, especially under the influence of EtOH.
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Consumo de Bebidas Alcohólicas , Etanol/toxicidad , Melanoma Experimental/patología , Neovascularización Patológica/inducido químicamente , Neoplasias Cutáneas/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Progresión de la Enfermedad , Endostatinas/análisis , Masculino , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Neoplasias Cutáneas/irrigación sanguínea , Neoplasias Cutáneas/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Molecular mechanisms of salt-sensitive (SS) hypertension related to renal inflammation have not been defined. We seek to determine whether a high-salt (HS) diet induces renal activation of NF-kappaB and upregulation of TNF-alpha related to the development of hypertension in Dahl SS rats. Six 8-wk-old male Dahl SS rats received a HS diet (4%), and six Dahl SS rats received a low-sodium diet (LS, 0.3%) for 5 wk. In the end, mean arterial pressure was determined in conscious rats by continuous monitoring through a catheter placed in the carotid artery. Mean arterial pressure was significantly higher in the HS than the LS group (177.9 +/- 3.7 vs. 109.4 +/- 2.9 mmHg, P < 0.001). There was a significant increase in urinary albumin secretion in the HS group compared with the LS group (22.3 +/- 2.6 vs. 6.1 +/- 0.7 mg/day; P < 0.001). Electrophoretic mobility shift assay demonstrated that the binding activity of NF-kappaB p65 proteins in the kidneys of Dahl SS rats was significantly increased by 53% in the HS group compared with the LS group (P = 0.007). ELISA indicated that renal protein levels of TNF-alpha, but not IL-6, interferon-gamma, and CCL28, were significantly higher in the HS than the LS group (2.3 +/- 0.8 vs. 0.7 +/- 0.2 pg/mg; P = 0.036). We demonstrated that plasma levels of TNF-alpha were significantly increased by fivefold in Dahl SS rats on a HS diet compared with a LS diet. Also, we found that increased physiologically relevant sodium concentration (10 mmol/l) directly stimulated NF-kappaB activation in cultured human renal proximal tubular epithelial cells. These findings support the hypothesis that activation of NF-kappaB and upregulation of TNF-alpha are the important renal mechanisms linking proinflammatory response to SS hypertension.
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Hipertensión Renal/metabolismo , FN-kappa B/metabolismo , Nefritis/metabolismo , Cloruro de Sodio Dietético , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Hipertensión Renal/inducido químicamente , Hipertensión Renal/complicaciones , Masculino , Nefritis/inducido químicamente , Nefritis/etiología , Ratas , Ratas Endogámicas Dahl , Estadística como Asunto , Activación Transcripcional , Regulación hacia ArribaRESUMEN
The role of angiostatic factors, including endostatin, in regulating physiological angiogenesis is poorly understood. We used normal adult rats under physiological resting conditions to examine the relationship between tissue endostatin, VEGF, and capillary density (CD) in the heart (high metabolic activity) versus the skeletal muscle (relatively low metabolic activity). The heart (left ventricle, LV) and skeletal muscle (anterior tibialis, AT) were dissected from 12-week-old male Sprague-Dawley rats. Transverse cryosections of LV and AT were stained with FITC-conjugated GS-I-lectin. CD was determined by analysis of randomly acquired digital images of the cryosections using Optimas software. Tissue protein levels of endostatin and VEGF were determined by ELISA assays. Tissue endostatin levels were lower in the LV and higher in the AT (135 +/- 39 vs. 663 +/- 114 pg/mg) and VEGF levels were higher in the LV and lower in the AT (41 +/- 3 vs. 27 +/- 4 pg/mg), respectively (n = 7, P < 0.01). CD in LV and AT were 3632 +/- 428 and 437 +/- 44/mm2, respectively (P < 0.01). We demonstrated that an 8.3-fold greater capillary density is related to a 4.9-fold lower level of tissue endostatin and a 1.5-fold higher level of tissue VEGF in the heart (LV) versus the skeletal muscle (AT) of normal rats under physiological resting conditions. Also, exercise training increased capillary density, decreased tissue endostatin and increased tissue VEGF in the skeletal muscle (AT). These findings suggest that tissue endostatin content correlates inversely with capillary network in the muscle tissues with different metabolic activity, and that tissue endostatin may play a very important role in the metabolic control of angiogenesis under physiological conditions.
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Capilares/crecimiento & desarrollo , Endostatinas/fisiología , Ventrículos Cardíacos/química , Músculo Esquelético/química , Animales , Endostatinas/análisis , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Lectinas/metabolismo , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Physical inactivity increases the risk of cancer and atherosclerosis; the impaired regulation of angiogenesis is often associated with the development of these diseases. We hypothesize that exercise increases circulating sFlt-1, an endogenous VEGF inhibitor, which may functionally decrease plasma levels of free VEGF. MATERIAL/METHODS: 5 healthy male adults were assigned to a treadmill exercise study. The peak speed and the time spent at peak speed on the treadmill were 4.8+/-1.0 miles/h and 6.8+/-2.6 minutes, respectively. Plasma levels of sFlt-1 and VEGF were determined using ELISA (R&D Systems). RESULTS: Basal plasma levels of sFlt-1 (before exercise) were 48.8+/-9.0 pg/ml. Plasma levels of sFlt-1 increased to 72.9+/-14.6 pg/ml at 0.5 h after exercise, compared to the basal levels (49% higher, P=0.0048). The plasma levels then returned to 47.5+/-14.3 and 43.3+/-10.2 pg/ml, at 2 and 6 h after exercise, respectively. There was a significant positive correlation between % increase in plasma levels of sFlt-1 and total peak oxygen consumption during exercise (R2=0.8244; P<0.01). Basal plasma levels of unbound VEGF were 37.3+/-7.7 pg/ml, then decreased to 28.2+/-6.3, 17.5+/-2.5, and 26.6+/-6.4 pg/ml, at 0.5, 2, and 6 h, respectively, after exercise. There was a significant increase in basal plasma levels of sFlt-1 after repeated exercise for 2 weeks. CONCLUSIONS: We have demonstrated that circulating sFlt-1 is significantly increased by exercise in healthy volunteers, which is functionally associated with a transient decrease in circulating free VEGF. This data may provide new insight into the molecular links between physical inactivity and risk of cancer and atherosclerosis.