RESUMEN
AIM: To study whether microvascular leukocyte accumulation after rat renal ischemia and reperfusion (IR) is decreased by Rho kinase inhibition, independently of effects upon nitric oxide (NO) and renal blood flow. METHODS: Male Wistar rats were subjected to 60 min of ischemia by bilateral clamping and 60 min of reperfusion of the renal arteries, or a sham procedure. Haemodynamics were monitored and microsphere blood flow to the kidneys was measured. The infusion of the Rho kinase inhibitor (Y27632) was commenced before clamping and IR. The NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), was administered after the start of reperfusion whilst the dopamine-1 receptor agonist fenoldopam, a renal vasodilator, was infused during the reperfusion period. Digital imaging microscopy analysis of cryosections was done to determine leukocyte accumulation and vasodilator-stimulated phosphoprotein serine 239 phosphorylation (p-VASP ser 239), a marker of endothelial NO. RESULTS: Leukocytes (60-70% neutrophils) accumulated within blood vessels in the corticomedullary junction and medulla of the kidney. Leukocyte accumulation was markedly reduced by the Rho kinase inhibitor but not by fenoldopam. However, both drugs improved renal blood flow and microvascular expression of p-VASP ser 239 in the corticomedullary junction and medulla, which were decreased following IR. L-NAME treatment of IR animals pretreated with the Rho kinase inhibitor reduced blood flow and p-VASP ser 239 expression and increased leukocyte accumulation. CONCLUSION: Early microvascular leukocyte accumulation in the corticomedullary junction and medulla of the rat kidney after IR is ameliorated by Rho kinase inhibition. This effect is partly independent upon attenuation of decreased NO and renal blood flow.
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Riñón/enzimología , Leucocitos/enzimología , Microcirculación/fisiología , Óxido Nítrico/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Daño por Reperfusión/enzimología , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/fisiología , Amidas/farmacología , Amidas/uso terapéutico , Animales , Riñón/irrigación sanguínea , Leucocitos/efectos de los fármacos , Leucocitos/patología , Masculino , Microcirculación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Ratas , Ratas Wistar , Circulación Renal/efectos de los fármacos , Circulación Renal/fisiología , Daño por Reperfusión/tratamiento farmacológicoRESUMEN
BACKGROUND/AIMS: Decreased endothelium-dependent vasodilation and blood flow in renal ischemia-reperfusion (IR) may result in part from rho-kinase activation, and cyclooxygenase (COX) activation, and resultant reactive oxygen species (ROS) may be involved. METHODS: We tested this hypothesis in male Wistar rats, subjected to 60 min of bilateral clamping of the renal arteries and 60 min of reperfusion or a sham procedure, and treated by the rho-kinase inhibitor Y27632 (1 mg/kg) and/or the nonspecific COX inhibitor diclofenac (10 mg/kg). Renal blood flow was measured by fluorescent microspheres, and ROS in the arterial endothelium was quantified by dihydroethidium staining. Endothelium-dependent vasodilation was determined by an acetylcholine concentration-response curve in the presence or absence of diclofenac (10 microM). RESULTS: Y27632 increased renal blood flow and reduced ROS in vivo, and improved endothelium-dependent vasodilation in vitro, following IR with or without diclofenac. Following IR, diclofenac had no effect on renal blood flow and ROS in vivo, but improved endothelium-dependent vasodilation in vitro. CONCLUSION: Activation of rho-kinase impairs endothelium-dependent vasodilation and perfusion following renal IR, independently of COX and resultant ROS. In contrast, the vasodilatory effect of rho-kinase inhibition may be partly mediated by decreasing ROS, unrelated to COX and resultant vasoconstricting prostanoids.
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Endotelio Vascular/metabolismo , Enfermedades Renales/metabolismo , Riñón/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/metabolismo , Quinasas Asociadas a rho/metabolismo , Animales , Velocidad del Flujo Sanguíneo , Riñón/irrigación sanguínea , Masculino , Ratas , Ratas Wistar , Transducción de Señal , VasodilataciónRESUMEN
Pulmonary arterial hypertension (PAH) is often treated with endothelin (ET) receptor blockade or phosphodiesterase-5 (PDE5) inhibition. Little is known about the specific effects on right ventricular (RV) function and metabolism. We determined the effects of single and combination treatment with Bosentan [an ET type A (ET(A))/type B (ET(B)) receptor blocker] and Sildenafil (a PDE5 inhibitor) on RV function and oxidative metabolism in monocrotaline (MCT)-induced PAH. Fourteen days after MCT injection, male Wistar rats were orally treated for 10 days with Bosentan, Sildenafil, or both. RV catheterization and echocardiography showed that MCT clearly induced PAH. This was evidenced by increased RV systolic pressure, reduced cardiac output, increased pulmonary vascular resistance (PVR), and reduced RV fractional shortening. Quantitative histochemistry showed marked RV hypertrophy and fibrosis. Monotreatment with Bosentan or Sildenafil had no effect on RV systolic pressure or cardiac function, but RV fibrosis was reduced and RV capillarization increased. Combination treatment did not reduce RV systolic pressure, but significantly lowered PVR, and normalized cardiac output, RV fractional shortening, and fibrosis. Only combination treatment increased the mitochondrial capacity of the RV, as reflected by increased succinate dehydrogenase and cytochrome c oxidase activities, associated with an activation of PKG, as indicated by increased VASP phosphorylation. Moreover, significant interactions were found between Bosentan and Sildenafil on PVR, cardiac output, RV contractility, PKG activity, and mitochondrial capacity. These data indicate that the combination of Bosentan and Sildenafil may beneficially contribute to RV adaptation in PAH, not only by reducing PVR but also by acting on the mitochondria in the heart.
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Antagonistas de los Receptores de Endotelina , Hipertensión Pulmonar/fisiopatología , Mitocondrias Cardíacas/efectos de los fármacos , Inhibidores de Fosfodiesterasa 5 , Inhibidores de Fosfodiesterasa/farmacología , Arteria Pulmonar/fisiopatología , Animales , Peso Corporal/fisiología , Bosentán , Capilares/fisiología , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Difusión , Fibrosis , Hemodinámica/fisiología , Hipertensión Pulmonar/diagnóstico por imagen , Técnicas In Vitro , Masculino , Mitocondrias Cardíacas/enzimología , Contracción Miocárdica/efectos de los fármacos , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Mioglobina/metabolismo , Tamaño de los Órganos/fisiología , Consumo de Oxígeno , Piperazinas/farmacología , Purinas/farmacología , Ratas , Ratas Wistar , Citrato de Sildenafil , Sulfonamidas/farmacología , Sulfonas/farmacología , UltrasonografíaRESUMEN
Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4- and 70-kDa dextrans through the cytosol, and localization of 155- and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4- to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size.
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Caveolas/metabolismo , Dextranos/metabolismo , Sistemas de Liberación de Medicamentos , Endocitosis , Células Endoteliales/metabolismo , Colorantes Fluorescentes/metabolismo , Microburbujas , Ultrasonido , Adenosina Trifosfato/metabolismo , Androstadienos/farmacología , Animales , Transporte Biológico , Bovinos , Caveolina 1/metabolismo , Células Cultivadas , Clorpromazina/farmacología , Clatrina/metabolismo , Medios de Contraste/administración & dosificación , Citosol/metabolismo , Dextranos/administración & dosificación , Dextranos/química , Endocitosis/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Arteria Femoral/metabolismo , Filipina/farmacología , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/química , Imagenología Tridimensional , Infusiones Intravenosas , Microscopía Fluorescente , Peso Molecular , Fosfolípidos/administración & dosificación , Pinocitosis , Presión , Ratas , Ratas Wistar , Hexafluoruro de Azufre/administración & dosificación , Factores de Tiempo , Vesículas Transportadoras/metabolismo , WortmaninaRESUMEN
INTRODUCTION: The mechanisms by which mechanical ventilation (MV) can injure remote organs, such as the kidney, remain poorly understood. We hypothesized that upregulation of systemic inflammation, as reflected by plasma interleukin-6 (IL-6) levels, in response to a lung-injurious ventilatory strategy, ultimately results in kidney dysfunction mediated by local endothelin-1 (ET-1) production and renal vasoconstriction. METHODS: Healthy, male Wistar rats were randomized to 1 of 2 MV settings (n=9 per group) and ventilated for 4 h. One group had a lung-protective setting using peak inspiratory pressure of 14 cm H2O and a positive end-expiratory pressure of 5 cm H2O; the other had a lung-injurious strategy using a peak inspiratory pressure of 20 cm H2O and positive end-expiratory pressure of 2 cm H2O. Nine randomly assigned rats served as nonventilated controls. We measured venous and arterial blood pressure and cardiac output (thermodilution method), renal blood flow (RBF) using fluorescent microspheres, and calculated creatinine clearance, urine flow, and fractional sodium excretion. Histological lung damage was assessed using hematoxylin-eosin staining. Renal ET-1 and plasma ET-1 and IL-6 concentrations were measured using enzyme-linked immunosorbent assays. RESULTS: Lung injury scores were higher after lung-injurious MV than after lung-protective ventilation or in sham controls. Lung-injurious MV resulted in significant production of renal ET-1 compared with the lung-protective and control groups. Simultaneously, RBF in the lung-injurious MV group was approximately 40% lower (P<0.05) than in the control group and 28% lower (P<0.05) than in the lung-protective group. Plasma ET-1 and IL-6 levels did not differ among the groups and systemic hemodynamics, such as cardiac output, were comparable. There was no effect on creatinine clearance, fractional sodium excretion, urine output, or kidney histology. CONCLUSIONS: Lung-injurious MV for 4 h in healthy rats results in significant production of renal ET-1 and in decreased RBF, independent of IL-6. Decreased RBF has no observable effect on kidney function or histology.
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Endotelina-1/biosíntesis , Riñón/metabolismo , Lesión Pulmonar , Circulación Renal , Respiración Artificial/efectos adversos , Animales , Creatinina/metabolismo , Diuresis , Interleucina-6/sangre , Enfermedades Renales/etiología , Pulmón/patología , Masculino , Ratas , Ratas Wistar , Sodio/metabolismoRESUMEN
Activation of cytoskeleton regulator Rho-kinase during ischemia-reperfusion (I/R) plays a major role in I/R injury and apoptosis. Since Rho-kinase is a negative regulator of the pro-survival phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, we hypothesized that inhibition of Rho-kinase can prevent I/R-induced endothelial cell apoptosis by maintaining PI3-kinase/Akt activity and that protective effects of Rho-kinase inhibition are facilitated by prevention of F-actin rearrangement. Human umbilical vein endothelial cells were subjected to 1 h of simulated ischemia and 1 or 24 h of simulated reperfusion after treatment with Rho-kinase inhibitor Y-27632, PI3-kinase inhibitor wortmannin, F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide. Intracellular ATP levels decreased following I/R. Y-27632 treatment reduced I/R-induced apoptosis by 31% (P < 0.01) and maintained Akt activity. Both effects were blocked by co-treatment with wortmannin. Y-27632 treatment prevented the formation of F-actin bundles during I/R. Similar results were observed with cytochalasinD treatment. In contrast, latrunculinA and jasplakinolide treatment did not prevent the formation of F-actin bundles during I/R and had no effect on I/R-induced apoptosis. Apoptosis and Akt activity were inversely correlated (R (2) = 0.68, P < 0.05). In conclusion, prevention of F-actin rearrangement by Rho-kinase inhibition or by cytochalasinD treatment attenuated I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity.
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Actinas/química , Apoptosis/efectos de los fármacos , Endotelio Vascular/citología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Daño por Reperfusión , Quinasas Asociadas a rho/fisiología , Actinas/efectos de los fármacos , Amidas/farmacología , Androstadienos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Células Cultivadas , Citocalasina D/farmacología , Depsipéptidos/farmacología , Endotelio Vascular/efectos de los fármacos , Humanos , Piridinas/farmacología , Daño por Reperfusión/patología , Tiazolidinas/farmacología , Wortmanina , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Acute renal failure during human sepsis is often nonoliguric. To study the underlying mechanisms, renal function was assessed in endotoxic and control male Wistar rats during and after saline loading and treatment with the selective V2 receptor agonist desmopressin. Escherichia coli endotoxin (dose, 8 mg/kg) was administered from time (t)=0 to t=60 min; saline loading (rate, 5 mL/100 g per hour) was administered from t=0 to t=120 min. Thereafter, half of each group received desmopressin (dose, 10 microg) for 1 h. The inner medullary (IM) osmolality, hematocrit, plasma, and urinary concentrations of sodium, potassium, urea, and osmolality were measured; then, aquaporin 2 (AQP2) immunohistochemistry was performed. Plasma vasopressin concentrations were measured at t=180 min. Saline loading increased urine volume in all rats. In the endotoxic group, mean arterial pressure decreased when saline loading was stopped. Despite increased hematocrit and vasopressin levels (>16 pg/mL), the endotoxin group had a low IM osmolality (mean +/- SEM, 412+/-0.04 mOsm/kg H2O) in comparison with the control group (mean +/- SEM, 1,094+/-0.17 mOsm/kg H2O) and was not able to either decrease urine volume or raise urine osmolality. Desmopressin treatment in endotoxin-treated rats maintained mean arterial pressure, increased sodium reabsorption, IM osmolality, and urine osmolality, and decreased urine flow. The AQP2 intensity decreased in the endotoxin group, and the apical localization disappeared; both were not affected by desmopressin. Our results indicate that endotoxemia in rats acutely diminishes renal urinary concentration capacity and is associated with a decreased IM osmolality and diminished apical AQP2 localization. These findings may help to explain nonoliguric acute renal failure in human septic shock.
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Desamino Arginina Vasopresina/farmacología , Endotoxemia/tratamiento farmacológico , Animales , Acuaporina 2/análisis , Acuaporina 2/metabolismo , Desamino Arginina Vasopresina/administración & dosificación , Endotoxemia/inducido químicamente , Endotoxinas/administración & dosificación , Endotoxinas/farmacología , Hemodinámica/efectos de los fármacos , Inmunohistoquímica , Médula Renal/efectos de los fármacos , Médula Renal/metabolismo , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/metabolismo , Masculino , Concentración Osmolar , Potasio/metabolismo , Ratas , Ratas Wistar , Sodio/metabolismo , Vasopresinas/sangreRESUMEN
BACKGROUND: Damaged and/or dysfunctional microvascular endothelium has been implicated in the pathogenesis of ischemic acute renal failure (ARF). Rapidly occurring changes in the endothelial F-actin cytoskeleton as observed in vitro might be responsible, but have been proven difficult to measure accurately in situ. Therefore, the purpose of this study was to examine several methods of digital image analysis in order to quantify the alterations of endothelial F-actin after renal ischemia and reperfusion (I/R), and to relate these to deterioration of renal function. METHODS: Frozen sections of Sham and I/R rat kidneys were fixed in 4% formaldehyde and stained with rhodamine-phalloïdin. Microvascular structures were captured using a 3i Marianastrade mark digital imaging fluorescence microscope workstation. Images were analyzed using 3i SlideBooktrade mark software, employing several masking techniques and line-scans. RESULTS: Digital image analysis demonstrated a decrease in the mean intensity of rhodamine-phalloïdin fluorescence after I/R from 1030 +/- 187 to 735 +/- 121 a.u. (arbitrary units, mean +/- SD, n = 7). The number of F-actin fragments per pixel increased from (15.8 +/- 4.9) x 10(-5) to (20.7 +/- 3.5) x 10(-5) (n = 7), indicating cytoskeletal fragmentation. In addition, line-scan analysis demonstrated a disturbed spatial orientation of the F-actin cytoskeleton after I/R. Finally, the loss of F-actin correlated with a rise in plasma creatinine. CONCLUSIONS: The methods of digital image analysis described in the present study demonstrate that renal I/R induces profound changes in the F-actin cytoskeletal structure of microvascular endothelial cells, implicating an injured and dysfunctional microvascular endothelium, which may contribute to acute renal failure (ARF).
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Actinas/ultraestructura , Citoesqueleto/ultraestructura , Células Endoteliales/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Isquemia , Circulación Renal , Reperfusión , Animales , Masculino , Ratas , Ratas Wistar , Circulación Renal/fisiologíaRESUMEN
Renal ischemia-reperfusion (I/R) results in vascular dysfunction characterized by a reduced endothelium-dependent vasodilatation and subsequently impaired blood flow. In this study, we investigated the role of Rho kinase in endothelial nitric oxide synthase (eNOS)-mediated regulation of renal blood flow and vasomotor tone in renal I/R. Male Wistar rats were subjected to 60-min bilateral clamping of the renal arteries or sham procedure. One hour before the clamping, the Rho kinase inhibitor Y27632 (1 mg/kg) was intravenously infused. After I/R, renal blood flow was measured using fluorescent microspheres. I/R resulted in a 62% decrease in renal blood flow. In contrast, the blood flow decrease in the group treated with the Rho kinase inhibitor (YI/R) was prevented. Endothelium-dependent vasodilatation of renal arcuate arteries to ACh was measured ex vivo in a pressure myograph. These experiments demonstrated that the in vivo treatment with the Rho kinase inhibitor prevented the decrease in the nitric oxide (NO)-mediated vasodilator response. In addition, after I/R renal interlobar arteries showed a decrease in phosphorylated eNOS and vasodilator-stimulated phosphoprotein, a marker for bioactive NO, which was attenuated by in vivo Rho kinase inhibition. These findings indicate that in vivo inhibition of Rho kinase in renal I/R preserves renal blood flow by improving eNOS function.
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Riñón/irrigación sanguínea , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Circulación Renal/fisiología , Daño por Reperfusión/enzimología , Acetilcolina/sangre , Animales , Moléculas de Adhesión Celular/metabolismo , Hemodinámica , Péptidos y Proteínas de Señalización Intracelular , Riñón/enzimología , Masculino , Proteínas de Microfilamentos/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Norepinefrina/sangre , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ratas , Ratas Wistar , Arteria Renal/enzimología , Daño por Reperfusión/fisiopatología , Quinasas Asociadas a rhoRESUMEN
Recent evidence suggests that oxidative stress is a common denominator in many aspects of cardiovascular pathogenesis. Free cellular iron plays a crucial catalytic role in the formation of highly toxic hydroxyl radicals, and thereby it may aggravate the contribution of oxidative stress to cardiovascular disease. Therefore, iron chelation may be an effective therapeutic approach, but the progress in this area is hindered by the lack of effective agents. In this study, using the rat heart myoblast-derived cell line H9c2, we aimed to investigate whether the novel lipophilic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) protects the cells against hydrogen peroxide (H2O2)-induced cytotoxicity. Exposure of cells to 100 micromol/l H2O2 has within 4 h induced a complete dissipation of their mitochondrial membrane potential (DeltaPsim). Co-treatment with SIH dose-dependently reduced (EC50=0.8 micromol/l) or even completely abolished (3 micromol/l) this collapse. Furthermore, the latter SIH concentration was capable to fully prevent alterations in cell morphology, and inhibited both apoptosis (annexin-V staining, nuclear chromatin shrinkage, TUNEL positivity) and necrosis (propidium iodide staining), even 24 h after the H2O2 exposure. In comparison, deferoxamin (a commercially available hydrophilic iron chelator used in clinical practice and most previous studies) was cytoprotective only at three-order higher and clinically unachievable concentrations (EC50=1300 micromol/l). Thus, in this study, we present iron chelation as a very powerful tool by which oxidative stress-induced myocardial damage can be prevented.