RESUMEN
Optical coherence tomography angiography (OCTA) provides three-dimensional structural and semiquantitative imaging of microvasculature in vivo. We developed an OCTA imaging protocol for a murine kidney ischemia-reperfusion injury (IRI) model to investigate the correlation between renal microvascular changes and ischemic damage. Mice were divided into mild and moderate IRI groups according to the duration of ischemia (10 and 35 mins, respectively). Each animal was imaged at baseline; during ischemia; and at 1, 15, 30, 45, and 60 mins after ischemia. Amplitude decorrelation OCTA images were constructed with 1.5-, 3.0-, and 5.8-ms interscan times, to calculate the semiquantitative flow index in the superficial (50-70 µm) and the deep (220-340 µm) capillaries of the renal cortex. The mild IRI group showed no significant flow index change in both the superfial and the deep layers. The moderate IRI group showed a significantly decreased flow index from 15 and 45 mins in the superficial and deep layers, respectively. Seven weeks after IRI induction, the moderate IRI group showed lower kidney function and higher collagen deposition than the mild IRI group. OCTA imaging of the murine IRI model revealed changes in superficial blood flow after ischemic injury. A more pronounced decrease in superficial blood flow than in deep blood flow was associated with sustained dysfunction after IRI. Further investigation on post-IRI renal microvascular response using OCTA may improve our understanding of the relationship between the degree of ischemic insult and kidney function.
Asunto(s)
Daño por Reperfusión , Tomografía de Coherencia Óptica , Ratones , Animales , Riñón/diagnóstico por imagen , Riñón/irrigación sanguínea , Daño por Reperfusión/diagnóstico por imagen , Daño por Reperfusión/complicaciones , Isquemia/diagnóstico por imagen , Isquemia/complicaciones , Microvasos/diagnóstico por imagen , AngiografíaRESUMEN
BACKGROUND: ML171 is a potent nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor with isoform selectivity only for NOX1. This study is aimed at investigating the safety of ML171 after a single intraperitoneal (IP) injection in mice. METHODS: The toxicity of a single dose of ML171 was evaluated in 6-week-old Institute of Cancer Research (ICR) mice in a good laboratory practice (GLP) laboratory. Twenty-five mice of each sex were assigned to five groups: negative control, vehicle control, and 125, 250, and 500 mg/kg of ML171. All mice were acclimatized for one week before beginning the study. Mice received an IP injection of ML171 or vehicle. The general condition and mortality of the animals were observed. The mice were sacrificed to evaluate histopathology 14 days after the administration of ML171 or vehicle. RESULTS: Bodyweights were not significantly different in any group. Three males and one female died due to ML171 administration in the 500 mg/kg dose group. Autopsies of the surviving mice did not reveal any significant abnormalities after the injection of 125 mg/kg of ML171. However, the anterior lobe edge of the liver was thickened and adhesions between the liver and adjacent organs were observed in mice treated with 250 or 500 mg/kg of ML171. In addition, hypertrophy of centrilobular hepatocytes and inflammatory cell infiltration were observed after injection of 250 and 500 mg/kg of ML171. CONCLUSION: Our results indicate that the lethal IP injection dose of ML171 is 500 mg/kg for both males and females. Mortality were not observed for lower doses of ML171. The safe dose of single IP ML171 in ICR mice was 250 mg/kg or less. Further studies are needed to confirm the safety of ML171 in the human body.
Asunto(s)
NADPH Oxidasa 1/antagonistas & inhibidores , Fenotiazinas/farmacología , Fenotiazinas/toxicidad , Animales , Evaluación Preclínica de Medicamentos , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Isoformas de Proteínas , Pruebas de ToxicidadRESUMEN
The protective effects of alpha-1 antitrypsin (AAT) in tacrolimus (TAC)-induced renal injury was evaluated in a rat model. The TAC group rats were subcutaneously injected with 2 mg/kg TAC every day for four weeks. The TAC with AAT group was cotreated with daily subcutaneous injections of TAC and intraperitoneal injections of AAT (80 mg/kg) for four weeks. The effects of AAT on TAC-induced renal injury were evaluated using serum biochemistry, histopathology, and Western blotting. The TAC injection significantly increased renal interstitial fibrosis, inflammation, and apoptosis as compared to the control treatment. The histopathological examination showed that cotreatment of TAC and AAT attenuated interstitial fibrosis (collagen, fibronectin, and α-SMA staining), and α-SMA expression in Western blotting was also decreased. Immunohistochemical staining for inflammation (osteopontin and ED-1 staining) revealed improved interstitial inflammation in the TAC with AAT group compared to that in the TAC group. The TAC treatment increased renal apoptosis compared to the control treatment, based on the results of increased immunohistochemical staining of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), increased caspase-3 activity, and lower Bcl-2 to Bad expression ratio. However, AAT cotreatment significantly changed these markers and consequently showed decreased apoptosis. AAT protects against TAC-induced renal injury via antifibrotic, anti-inflammatory, and antiapoptotic effects.
Asunto(s)
Lesión Renal Aguda , Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Tacrolimus/efectos adversos , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Lesión Renal Aguda/prevención & control , Animales , Fibrosis , Masculino , Ratas , Ratas Sprague-Dawley , Tacrolimus/farmacología , alfa 1-AntitripsinaRESUMEN
The protective effects of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) 1 inhibition against kidney ischemia-reperfusion injury (IRI) remain uncertain. The bilateral kidney pedicles of C57BL/6 mice were clamped for 30 min to induce IRI. Madin-Darby Canine Kidney (MDCK) cells were incubated with H2O2 (1.4 mM) for 1 h to induce oxidative stress. ML171, a selective NOX1 inhibitor, and siRNA against NOX1 were treated to inhibit NOX1. NOX expression, oxidative stress, apoptosis assay, and mitogen-activated protein kinase (MAPK) pathway were evaluated. The kidney function deteriorated and the production of reactive oxygen species (ROS), including intracellular H2O2 production, increased due to IRI, whereas IRI-mediated kidney dysfunction and ROS generation were significantly attenuated by ML171. H2O2 evoked the changes in oxidative stress enzymes such as SOD2 and GPX in MDCK cells, which was mitigated by ML171. Treatment with ML171 and transfection with siRNA against NOX1 decreased the upregulation of NOX1 and NOX4 induced by H2O2 in MDCK cells. ML171 decreased caspase-3 activity, the Bcl-2/Bax ratio, and TUNEL-positive tubule cells in IRI mice and H2O2-treated MDCK cells. Among the MAPK pathways, ML171 affected ERK signaling by ERK phosphorylation in kidney tissues and tubular cells. NOX1-selective inhibition attenuated kidney IRI via inhibition of ROS-mediated ERK signaling.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Enfermedades Renales/enzimología , Riñón/enzimología , Sistema de Señalización de MAP Quinasas , NADPH Oxidasa 1/metabolismo , Daño por Reperfusión/enzimología , Animales , Perros , Riñón/patología , Enfermedades Renales/patología , Células de Riñón Canino Madin Darby , Masculino , Ratones , Daño por Reperfusión/patologíaRESUMEN
Fimasartan, a new angiotensin II receptor antagonist, reduces myocyte damage and stabilizes atherosclerotic plaque through its anti-inflammatory effect in animal studies. We investigated the protective effects of pretreatment with fimasartan on ischemia-reperfusion injury (IRI) in a mouse model of ischemic renal damage. C57BL/6 mice were pretreated with or without 5 (IR-F5) or 10 (IR-F10) mg/kg/day fimasartan for 3 days. Renal ischemia was induced by clamping bilateral renal vascular pedicles for 30 min. Histology, pro-inflammatory cytokines, and apoptosis assays were evaluated 24 h after IRI. Compared to the untreated group, blood urea nitrogen and serum creatinine levels were significantly lower in the IR-F10 group. IR-F10 kidneys showed less tubular necrosis and interstitial fibrosis than untreated kidneys. The expression of F4/80, a macrophage infiltration marker, and tumor necrosis factor (TNF)-α, decreased in the IR-F10 group. High-dose fimasartan treatment attenuated the upregulation of TNF-α, interleukin (IL)-1ß, and IL-6 in ischemic kidneys. Fewer TUNEL positive cells were observed in IR-F10 compared to control mice. Fimasartan caused a significant decrease in caspase-3 activity and the level of Bax, and increased the Bcl-2 level. Fimasartan preserved renal function and tubular architecture from IRI in a mouse ischemic renal injury model. Fimasartan also attenuated upregulation of inflammatory cytokines and decreased apoptosis of renal tubular cells. Our results suggest that fimasartan inhibited the process of tubular injury by preventing apoptosis induced by the inflammatory pathway.
RESUMEN
Contrast-induced acute kidney injury (CIAKI) is a leading cause of acute kidney injury following radiographic procedures. Intrarenal oxidative stress plays a critical role in CIAKI. Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidases (Noxs) are important sources of reactive oxygen species (ROS). Among the various types of Noxs, Nox4 is expressed predominantly in the kidney in rodents. Here, we evaluated the role of Nox4 and benefit of Nox4 inhibition on CIAKI using in vivo and in vitro models. HK-2 cells were treated with iohexol, with or without Nox4 knockdown, or the most specific Nox1/4 inhibitor (GKT137831). Effects of Nox4 inhibition on CIAKI mice were examined. Expression of Nox4 in HK-2 cells was significantly increased following iohexol exposure. Silencing of Nox4 rescued the production of ROS, downregulated pro-inflammatory markers (particularly phospho-p38) implicated in CIAKI, and reduced Bax and caspase 3/7 activity, which resulted in increased cellular survival in iohexol-treated HK-2 cells. Pretreatment with GKT137831 replicated these effects by decreasing levels of phospho-p38. In a CIAKI mouse model, even though the improvement of plasma blood urea nitrogen was unclear, pretreatment with GKT137831 resulted in preserved structure, reduced expression of 8-hydroxy-2'-deoxyguanosine (8OHdG) and kidney injury molecule-1 (KIM-1), and reduced number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive cells. These results suggest Nox4 as a key source of reactive oxygen species responsible for CIAKI and provide a novel potential option for prevention of CIAKI.
Asunto(s)
Lesión Renal Aguda/metabolismo , Medios de Contraste/efectos adversos , NADPH Oxidasa 4/metabolismo , Estrés Oxidativo , Lesión Renal Aguda/inducido químicamente , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Activación Enzimática , Silenciador del Gen , Humanos , Yohexol/farmacología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasa 4/genética , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Superóxidos/metabolismoRESUMEN
Although dipeptidyl peptidase-4 inhibitors, a class of antidiabetic drugs, have various pleiotropic effects, it remains undetermined whether gemigliptin has a beneficial effect on vascular calcification. Therefore, this study was performed to evaluate the effect of gemigliptin on vascular calcification in a rat model of adenine-induced chronic kidney disease and in cultured vascular smooth muscle cells. Gemigliptin attenuated calcification of abdominal aorta and expression of RUNX2 in adenine-induced chronic kidney disease rats. In cultured vascular smooth muscle cells, phosphate-induced increase in calcium content was reduced by gemigliptin. Gemigliptin reduced phosphate-induced PiT-1 mRNA expression, reactive oxygen species generation, and NADPH oxidase mRNA expression (p22phox and NOX4). The reduction of oxidative stress by gemigliptin was associated with the downregulation of phospho-PI3K/AKT expression. High phosphate increased the expression of frizzled-3 (FDZ3) and decreased the expression of dickkopf-related protein-1 (DKK-1) in the Wnt pathway. These changes were attenuated by gemigliptin treatment. Gemigliptin restored the decreased expression of vascular smooth muscle cells markers (α-SMA and SM22α) and increased expression of osteogenic makers (CBFA1, OSX, E11, and SOST) induced by phosphate. In conclusion, gemigliptin attenuated vascular calcification and osteogenic trans-differentiation in vascular smooth muscle cells via multiple steps including downregulation of PiT-1 expression and suppression of reactive oxygen species generation, phospho-PI3K/AKT, and the Wnt signaling pathway.
Asunto(s)
Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Piperidonas/farmacología , Pirimidinas/farmacología , Insuficiencia Renal Crónica/genética , Factor de Transcripción Pit-1/genética , Calcificación Vascular/prevención & control , Adenina , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/metabolismo , Aorta Abdominal/patología , Calcio/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , NADPH Oxidasa 4 , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Fosfatos/antagonistas & inhibidores , Fosfatos/farmacología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Insuficiencia Renal Crónica/inducido químicamente , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Factor de Transcripción Pit-1/antagonistas & inhibidores , Factor de Transcripción Pit-1/metabolismo , Calcificación Vascular/genética , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Vía de Señalización WntRESUMEN
Alpha1-antitrypsin (AAT) exerts its anti-inflammatory effect through regulating the activity of serine proteinases. This study evaluated the inhibitory effects of AAT against the transforming growth factor (TGF)-ß1 induced epithelial-to-mesenchymal transition (EMT) in unilateral ureter obstruction (UUO) mice and Madin-Darby canine kidney (MDCK) cells. C57BL/6 mice with induced UUO were injected intraperitoneally with AAT (80 mg/Kg) or vehicle for 7 days. MDCK cells were treated with TGF-ß1 (2 ng/mL) for 48 hours to induce EMT, and co-treated with AAT (10 mg/mL) to inhibit the EMT. Masson's trichrome and Sirius red staining was used to estimate the extent of renal fibrosis in UUO mice. The expression of alpha-smooth muscle actin (α-SMA), vimentin, fibronectin, collagen I, and E-cadherin in MDCK cells and kidney tissue were evaluated. Masson's and Sirius red staining revealed that the area of renal fibrosis was significantly smaller in AAT treated UUO group compared with that of UUO and vehicle treated UUO groups. AAT treatment attenuated upregulation of Smad2/3 phosphorylation in UUO mouse model. Co-treatment of MDCK cells with TGF-ß1 and AAT significantly attenuated the changes in the expression of α-SMA, vimentin, fibronectin, collagen I, and E-cadherin. AAT also decreased the phosphorylated Smad3 expression and the phosphorylated Smad3/Smad3 ratio in MDCK cells. AAT treatment inhibited EMT induced by TGF-ß1 in MDCK cells and attenuated renal fibrosis in the UUO mouse model. The results of this work suggest that AAT could inhibit the process of EMT through the suppression of TGF-ß/Smad3 signaling.
Asunto(s)
Transición Epitelial-Mesenquimal/efectos de los fármacos , Riñón/patología , Factor de Crecimiento Transformador beta1/farmacología , alfa 1-Antitripsina/uso terapéutico , Actinas/metabolismo , Animales , Biomarcadores/metabolismo , Cadherinas/metabolismo , Supervivencia Celular/efectos de los fármacos , Colágeno Tipo I/metabolismo , Perros , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fibronectinas/metabolismo , Fibrosis , Técnica del Anticuerpo Fluorescente , Células de Riñón Canino Madin Darby , Masculino , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Smad/metabolismo , Coloración y Etiquetado , Regulación hacia Arriba/efectos de los fármacos , Obstrucción Ureteral/tratamiento farmacológico , Obstrucción Ureteral/patología , Vimentina/metabolismoRESUMEN
Reactive oxygen species (ROS) generation during purine metabolism is associated with xanthine oxidase and uric acid. However, the direct effect of hypoxanthine on ROS generation and atherosclerosis has not been evaluated. Smoking and heavy drinking are associated with elevated levels of hypoxanthine. In this study, we investigated the role of hypoxanthine on cholesterol synthesis and atherosclerosis development, particularly in apolipoprotein E (APOE)-deficient mice. The effect of hypoxanthine on the regulation of cholesterol synthesis and atherosclerosis were evaluated in Apoe knockout (KO) mice and cultured HepG2 cells. Hypoxanthine markedly increased serum cholesterol levels and the atherosclerotic plaque area in Apoe KO mice. In HepG2 cells, hypoxanthine increased intracellular ROS production. Hypoxanthine increased cholesterol accumulation and decreased APOE and ATP-binding cassette transporter A1 (ABCA1) mRNA and protein expression in HepG2 cells. Furthermore, H2 O2 also increased cholesterol accumulation and decreased APOE and ABCA1 expression. This effect was partially reversible by treatment with the antioxidant N-acetyl cysteine and allopurinol. Hypoxanthine and APOE knockdown using APOE-siRNA synergistically induced cholesterol accumulation and reduced APOE and ABCA1 expression. Hypoxanthine induces cholesterol accumulation in hepatic cells through alterations in enzymes that control lipid transport and induces atherosclerosis in APOE-deficient cells and mice. These effects are partially mediated through ROS produced in response to hypoxanthine.
Asunto(s)
Apolipoproteínas E/deficiencia , Aterosclerosis/patología , Colesterol/metabolismo , Hipoxantina/farmacología , Acetilcisteína/farmacología , Alopurinol/farmacología , Animales , Apolipoproteínas E/metabolismo , Aterosclerosis/sangre , Colesterol/sangre , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Células Hep G2 , Humanos , Peróxido de Hidrógeno/toxicidad , Hipercolesterolemia/patología , Lipogénesis/efectos de los fármacos , Lipogénesis/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Placa Aterosclerótica/sangre , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Encapsulating peritoneal sclerosis (EPS) involves excessive peritoneal fibrosis in patients on peritoneal dialysis, eventually leading to visceral constriction and bowel obstruction. Few studies have investigated epigenetic mechanisms relating to EPS. Here we evaluated the therapeutic effects of DNA demethylation in experimental EPS. Experimental EPS was induced by intraperitoneal injection of 0.1% chlorhexidine gluconate (CG) and 15% ethanol in non-uremic male Sprague-Dawley (SD) rats. Rats were divided into three groups: group C (N=5) with saline injection only, group CG (N=7) with EPS induction for 4 weeks, and chlorhexidine gluconate and azacytidine (CGA) treated group (N=7) with EPS induction for 4 weeks and 5'-azacytidine injection for the last 2 weeks. Morphometric analysis of peritoneum and immunohistochemical staining for type 1 collagen and α-smooth muscle actin (α-SMA) were performed. Expressions of transforming growth factor-ß (TGF-ß), fibroblast-specific protein 1 (FSP1), and DNA methyltransferase 1 (DNMT1) were analyzed by Western blot. Methylation-specific polymerase chain reaction (PCR) for Ras GTPase activating-like protein 1 (RASAL1) was performed with measurement of RASAL1 protein expression. Parietal peritoneal thickness and the number of vessels in omental tissue were significantly decreased in group CGA compared to group CG, as were the expressions of type 1 collagen, α-SMA, TGF-ß, and FSP1. DNMT1 was significantly increased in group CG, and reduced in group CGA. RASAL1 hypermethylation was associated with decreased RASAL1 protein expression in group CG, which was reversed in group CGA. DNA demethylation by 5'-azacytidine treatment improved pathologic changes of the peritoneum in experimental EPS, and was associated with reversal of increased DNMT1 expression and RASAL1 hypermethylation.
Asunto(s)
Azacitidina/farmacología , Metilación de ADN , Diálisis Peritoneal/efectos adversos , Fibrosis Peritoneal/fisiopatología , Animales , Clorhexidina/análogos & derivados , Clorhexidina/toxicidad , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , Modelos Animales de Enfermedad , Etanol/toxicidad , Regulación de la Expresión Génica , Masculino , Fibrosis Peritoneal/etiología , Fibrosis Peritoneal/genética , Ratas , Ratas Sprague-Dawley , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
BACKGROUND: It has been previously demonstrated that listening to 1/f sound effectively reduces stress. However, these findings have been inconsistent and further study on the relationship between 1/f sound and the stress response is consequently necessary. OBJECTIVE: The present study examined whether sound with 1/f properties (1/f sound) affects stress-induced electroencephalogram (EEG) changes. METHODS: Twenty-six subjects who voluntarily participated in the study were randomly assigned to the experimental or control group. Data from four participants were excluded because of EEG artifacts. A mental arithmetic task was used as a stressor. Participants in the experiment group listened to 1/f sound for 5 minutes and 33 seconds, while participants in the control group sat quietly for the same duration. EEG recordings were obtained at various points throughout the experiment. After the experiment, participants completed a questionnaire on the affective impact of the 1/f sound. RESULTS: The results indicated that the mental arithmetic task effectively induced a stress response measurable by EEG. Relative theta power at all electrode sites was significantly lower than baseline in both the control and experimental group. Relative alpha power was significantly lower, and relative beta power was significantly higher in the T3 and T4 areas. Secondly, 1/f sound and simple resting affected task-associated EEG changes in a similar manner. Finally, participants reported in the questionnaire that they experienced a positive feeling in response to the 1/f sound. CONCLUSIONS: Our results suggest that a commercialized 1/f sound product is not more effective than simple resting in alleviating the physiological stress response.
Asunto(s)
Sonido , Estrés Psicológico/prevención & control , Electroencefalografía , Femenino , Humanos , MasculinoRESUMEN
BACKGROUND/AIMS: Circulatory asymmetric dimethylarginine (ADMA) is correlated with proteinuria and endothelial dysfunction in patients with proteinuric renal diseases. However, it is not known whether proteinuria itself affects expression of dimethylarginine dimethylaminohydrolase (DDAH), a degrading enzyme of ADMA, in kidney. The aim of this study is to evaluate the direct effects of losartan and/or pentoxifylline on expression of renal DDAH-1 and its relation to oxidative stress in the setting of albuminuria. METHODS: Using NRK52E cells, DDAH-1 mRNA and protein were determined after exposure to albumin with losartan and/or pentoxifylline. Reactive oxygen species (ROS), PKC activity, and NOX-4 mRNA were also measured. In addition, the effect of losartan and/or pentoxifylline on renal expression of DDAH-1 and serum ADMA were evaluated in a rat model of proteinuric nephropathy. RESULTS: Exposure to albumin resulted in increased release of N-acetyl-ß-D-glucosaminidase along with an increase of TNF-α, 8-hydroxy-2'-deoxyguanosine, and angiotensin II in NRK52E cells. Losartan and pentoxifylline reversed albumin-induced decrease of DDAH-1 mRNA and protein expression and DDAH-1 activity. The effects of losartan and pentoxifylline on DDAH-1 mRNA were associated with reduction of ROS. In addition, treatment with losartan and pentoxifylline resulted in an attenuated change of renal DDAH-1 protein expression and serum ADMA levels in vivo. CONCLUSION: DDAH-1 was positively regulated by losartan and pentoxifylline with its antioxidative effect in albumin-exposed renal proximal tubular cells. Combined treatment with losartan and pentoxifylline has a direct beneficial effect on expression of renal DDAH-1, and, thus, at least in part, modulates the circulatory levels of ADMA in proteinuric nephropathy.
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Albuminuria/enzimología , Amidohidrolasas/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Depuradores de Radicales Libres/uso terapéutico , Losartán/uso terapéutico , Pentoxifilina/uso terapéutico , Acetilglucosaminidasa/metabolismo , Albuminuria/tratamiento farmacológico , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Arginina/análogos & derivados , Arginina/metabolismo , Línea Celular , Depuradores de Radicales Libres/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/enzimología , Losartán/farmacología , Estrés Oxidativo/efectos de los fármacos , Pentoxifilina/farmacología , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Aquaporin 3 (AQP3) is expressed in many tissues including the peritoneum and kidney. In cultured mesothelial cells, glucose up-regulates AQP3, which may be important for water transport through the peritoneal membrane. However, there has been no research into the role of AQP3 in human peritoneal mesothelial cell (HPMC) migration or peritoneal fibrosis. We investigated the effects of transforming growth factor-ß1 (TGF-ß1) on AQP3 expression in HPMCs. We also investigated the role of AQP3 in the peritoneal wound healing process in rats. Chronic exposure to glucose-containing solution increased peritoneal myofibroblasts, with TGF-ß1 and AQP3 expression in a model of long-term peritoneal dialysis. In vitro, TGF-ß1 induced AQP3 expression in HPMCs. AQP3 knockdown by small-interfering RNA inhibited TGF-ß1-induced AQP3 and α-smooth muscle actin expression and also slowed HPMC migration. AQP3 overexpression induced faster migration of HPMCs. Treatment with an extracellular signal-regulated kinase inhibitor and p38 kinase inhibitor attenuated TGF-ß1-induced AQP3 expression in HPMCs. These data suggest that TGF-ß1 induces AQP3 and that AQP3 has a critical role in TGF-ß-induced HPMC migration. These findings provide evidence of a novel role for AQP3 in peritoneal fibrosis and wound healing. The effect of TGF-ß1 on AQP3 expression in HPMCs is mediated, at least in part, by ERK and p38 signaling.
Asunto(s)
Acuaporina 3/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Cavidad Peritoneal/patología , Factor de Crecimiento Transformador beta1/farmacología , Regulación hacia Arriba/genética , Cicatrización de Heridas/efectos de los fármacos , Actinas/metabolismo , Animales , Acuaporina 3/metabolismo , Biomarcadores/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Fibrosis , Técnicas de Silenciamiento del Gen , Glucosa/farmacología , Humanos , Masculino , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patología , Diálisis Peritoneal , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Soluciones , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: We investigated whether ex vivo mesothelial cells found in peritoneal dialysis (PD) effluents were representative of the in vivo epithelial-to-mesenchymal transition (EMT) in peritoneal membrane. METHODS: Thirty-six male Sprague-Dawley rats were equally divided into three groups: Group C (control), no PD; Group D, infused with 4.25% Dianeal and Group P, infused with 4.25% Physioneal. PD infusions (25 mL) were given twice daily for 8 weeks. The in vivo study included morphometric analyses performed on the peritoneal membranes of tissue specimens obtained at the end of the study. The ex vivo study included peritoneal mesothelial cells collected from PD effluent and cultured to confluence. Cells were scored with light microscopy. RESULTS: PD for 8 weeks induced significant EMT. The in vivo expression of EMT markers (α-smooth muscle actin:E-cadherin ratio, matrix metalloproteinase-2 and Snail) was higher in Group D than in Group P. However, ex vivo EMT marker expression was similar in cells derived from Groups D and P. A significant correlation was observed among in vivo EMT markers. Moreover, the ex vivo cell score increased with time on PD. However, changes in the ex vivo cell score did not correlated with changes in the in vivo EMT marker expression. Furthermore, we found no correlation between ex vivo and in vivo cells in the expression of EMT markers. CONCLUSIONS: In this animal study, ex vivo findings did not reflect the in vivo EMT changes in the peritoneum. It may be necessary to improve the current methodology for ex vivo studies.
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Soluciones para Diálisis/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Epitelio/patología , Diálisis Peritoneal , Peritoneo/patología , Actinas/metabolismo , Animales , Biomarcadores/metabolismo , Cadherinas/metabolismo , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Modelos Animales , Peritoneo/efectos de los fármacos , Peritoneo/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Transcripción de la Familia Snail , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Epithelial-mesenchymal transition (EMT) is important in the development of peritoneal fibrosis. Glucose degradation products (GDPs) may induce EMT in human peritoneal mesothelial cells (HPMCs). METHODS: The effects of individual GDPs and GDPs derived from peritoneal dialysis fluid (PDF) in both HPMCs and peritoneal membranes were evaluated. EMT was assessed with alpha-smooth muscle actin (alpha-SMA) and E-cadherin. RESULTS: In vitro, alpha-SMA protein and mRNA levels increased in the presence of the GDPs (formaldehyde, glyoxal, methylglyoxal, and 3-deoxyglucosone), and E-cadherin decreased. Changes in the EMT markers were most prominent after exposure to 3-deoxyglucosone. Changes in both alpha-SMA and E-cadherin protein levels were less with low (L)-GDP bicarbonate/lactate-buffered PDF compared to high (H)-GDP PDF. In the rat model after 8 weeks' PDF infusion, the alpha-SMA/E-cadherin mRNA ratio increased in the H-GDP group compared with the L-GDP group (p < 0.05). The peritoneum in the H-GDP group tended to be thicker (p = 0.052) and had more blood vessels than that in the L-GDP group (p < 0.05). Tissue staining for TGF-beta1 decreased in the L-GDP group. Dual-stained cytokeratin and alpha-SMA-positive myofibroblasts in the submesothelial layer were more prominent in the H-GDP group. CONCLUSION: GDPs found in PDF induce EMT of HPMCs, which is associated with peritoneal fibrosis and vascularization. Conversely, L-GDP PDF reduces EMT and peritoneal fibrosis.
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Bicarbonatos/farmacología , Transdiferenciación Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Glucosa/metabolismo , Soluciones para Hemodiálisis , Ácido Láctico/farmacología , Mesodermo/citología , Mesodermo/efectos de los fármacos , Mesodermo/fisiología , Peritoneo/citología , Peritoneo/efectos de los fármacos , Diálisis Renal , Animales , Células Cultivadas , Humanos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Three amino acid residues (His119, Glu164, and Glu338) in the active site of Thermus caldophilus GK24 beta- glycosidase (Tca beta-glycosidase), a family 1 glycosyl hydrolase, were mutated by site-directed mutagenesis. To verify the key catalytic residues, Glu164 and Glu338 were changed to Gly and Gln, respectively. The E164G mutation resulted in drastic reductions of both beta-galactosidase and beta-glucosidase activities, and the E338Q mutation caused complete loss of activity, confirming that the two residues are essential for the reaction process of glycosidic linkage hydrolysis. To investigate the role of His119 in substrate binding and enzyme activity, the residue was substituted with Gly. The H119G mutant showed 53-fold reduced activity on 5 mM p-nitrophenyl beta-Dgalactopyranoside, when compared with the wild type; however, both the wild-type and mutant enzymes showed similar activity on 5 mM p-nitrophenyl beta-D-glucopyranoside at 75degreeC. Kinetic analysis with p-nitrophenyl beta-D-galactopyranoside revealed that the kcat value of the H119G mutant was 76.3-fold lower than that of the wild type, but the Km of the mutant was 15.3-fold higher than that of the wild type owing to the much lower affinity of the mutant. Thus, the catalytic efficiency (kcat/Km) of the mutant decreased to 0.08% to that of the wild type. The kcat value of the H119G mutant for p-nitrophenyl beta- D-glucopyranoside was 5.1-fold higher than that of the wild type, but the catalytic efficiency of the mutant was 2.5% of that of the wild type. The H119G mutation gave rise to changes in optima pH (from 5.5-6.5 to 5.5) and temperature (from 90 degrees C to 80-85 degrees C). This difference of temperature optima originated in the decrease of H119G's thermostability. These results indicate that His119 is a crucial residue in beta- galactosidase and beta-glucosidase activities and also influences the enzyme's substrate binding affinity and thermostability.
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Mutación Missense , Thermus/enzimología , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , Thermus/química , Thermus/genética , beta-Galactosidasa/químicaRESUMEN
BACKGROUND: TGF-beta is involved in peritoneal changes during long-term peritoneal dialysis (PD). TGF-beta induces betaig-h3 in several cell lines, and betaig-h3 may be a marker for biologically active TGF-beta. However, no study has reported induction of betaig-h3 in human peritoneal mesothelial cells (HPMCs) or its involvement in PD-related peritoneal membrane changes. METHODS: We used cultured HPMCs to investigate the biological roles of betaig-h3 during mesothelial cell injury and repair, employing the adhesion, spreading, scratching and cell migration assays. Changes in betaig-h3 expression after high glucose exposure in vivo were also evaluated using an animal chronic PD model. RESULTS: In vitro, TGF-beta1 induced betaig-h3 in cultured HPMCs, and betaig-h3-mediated mesothelial cell adhesion occurred via alphavbeta3 integrin. betaig-h3 enhanced mesothelial cell adhesion and migration and, in part, wound healing during mesothelial cell injury. The animal study demonstrated that compared to the control group, betaig-h3 concentrations in the dialysate effluent increased in the dialysis group with alterations in peritoneal structure and function during PD, and betaig-h3 positively correlated with peritoneal solute transport. Immunohistochemical and immunoblotting results showed that betaig-h3 localizes in the mesothelium and submesothelial matrix of the parietal peritoneum, and in the vascular endothelium of omentum. betaig-h3 protein expression was higher in the dialysis group. CONCLUSION: In vitro, betaig-h3 induced by TGF-beta1 in HPMCs improved adhesion and migration of HPMCs during wound healing. In the chronic infusion model of PD, betaig-h3 played a role in the functional deterioration of the peritoneal membrane, which is associated with fibrosis.
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Proteínas de la Matriz Extracelular/fisiología , Diálisis Peritoneal , Factor de Crecimiento Transformador beta/fisiología , Células Cultivadas , Células Epiteliales/metabolismo , Proteínas de la Matriz Extracelular/biosíntesis , Humanos , Peritoneo/citología , Peritoneo/metabolismo , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
In the present study, we examined the effects of a new peritoneal dialysis fluid (PDF) with a low level of low glucose degradation products (GDP) on the functional and structural stability of the peritoneal membrane (PM). Male Sprague-Dawley rats were divided into three groups: group C (n = 8), without dialysate infusion; group P (n = 12), infused with low-level GDP solution (4.25% Physioneal, pH 7.0-7.4); and group D (n = 12), infused with conventional solution (4.25% Dianeal, pH 5.2, adjusted to pH 7.0). In groups D and P, animals were infused through a permanent catheter with 25 mL of PDF, twice daily for 8 weeks. Lipopolysaccharide was added into the PDF immediately before infusion on days 8, 9 and 10 in the two dialysis groups. When compared with group P, group D showed a higher glucose mass transfer at weeks 6 and 8, D/P urea at week 8, TGF-beta1 at weeks 4 and 8, and VEGF level at week 8. The submesothelial matrix layer of the parietal peritoneum was significantly thickened in group D and the lectin-stained blood vessels in this layer were well-visualized in group D compared with group P. There were significantly more peritoneal blood vessels in group D than group P. The transforming growth factor-beta induced gene-h3 (betaig-h3) and TGF-beta1 levels in the peritoneal effluent correlated with the submesothelial thickness, which correlated with the dialysate-to-plasma ratio (D/P) of protein and, inversely, with the rate of glucose transport (D/D(0) glucose, where D is glucose concentration in the dialysate and D(0) is glucose concentration in the dialysis solution before it is infused into the peritoneal cavity). The present study showed that low-GDP PDF effectively attenuated the peritoneal vascularization and fibrosis related to conventional solution.
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Soluciones para Diálisis/farmacología , Glucosa/farmacología , Diálisis Peritoneal , Peritoneo/efectos de los fármacos , Animales , Western Blotting , Proteínas de la Matriz Extracelular/metabolismo , Fibrosis , Inmunohistoquímica , Masculino , Peritoneo/irrigación sanguínea , Peritoneo/metabolismo , Peritoneo/patología , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Sympathetic nerves innervate the airways of most species but their reflex regulation has been essentially unstudied. Here we demonstrate sympathetic nerve-mediated reflex relaxation of airway smooth muscle measured in situ in the guinea-pig trachea. Retrograde tracing, immunohistochemistry and electrophysiological analysis identified a population of substance P-containing capsaicin-sensitive spinal afferent neurones in the upper thoracic (T1-T4) dorsal root ganglia (DRG) that innervate the airways and lung. After bilateral vagotomy, atropine pretreatment and pre-contraction of the trachealis with histamine, nebulized capsaicin (10-60 microm) evoked a 63+/-7% reversal of the histamine-induced contraction of the trachealis. Either the beta-adrenoceptor antagonist propranolol (2 microm, administered directly to the trachea) or bilateral sympathetic nerve denervation of the trachea essentially abolished these reflexes (10+/-9% and 6+/-4% relaxations, respectively), suggesting that they were mediated primarily, if not exclusively, by sympathetic adrenergic nerve activation. Cutting the upper thoracic dorsal roots carrying the central processes of airway spinal afferents also markedly blocked the relaxations (9+/-5% relaxation). Comparable inhibitory effects were observed following intravenous pretreatment with neurokinin receptor antagonists (3+/-7% relaxations). These reflexes were not accompanied by consistent changes in heart rate or blood pressure. By contrast, stimulating the rostral cut ends of the cervical vagus nerves also evoked a sympathetic adrenergic nerve-mediated relaxation that were accompanied by marked alterations in blood pressure. The results indicate that the capsaicin-induced reflex-mediated relaxation of airway smooth muscle following vagotomy is mediated by sequential activation of tachykinin-containing spinal afferent and sympathetic efferent nerves innervating airways. This sympathetic nerve-mediated response may serve to oppose airway contraction induced by parasympathetic nerve activation in the airways.
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Fibras Adrenérgicas/fisiología , Reflejo/fisiología , Tráquea/fisiología , Fibras Adrenérgicas/efectos de los fármacos , Animales , Capsaicina/farmacología , Relación Dosis-Respuesta a Droga , Cobayas , Masculino , Relajación Muscular/efectos de los fármacos , Relajación Muscular/fisiología , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Reflejo/efectos de los fármacos , Tráquea/efectos de los fármacosRESUMEN
Astrocytes promote the formation and function of excitatory synapses in the CNS. However, whether and how astrocytes modulate inhibitory synaptogenesis are essentially unknown. We asked whether astrocytes regulate the formation of inhibitory synapses between hippocampal neurons during maturation in vitro. Neuronal coculture with astrocytes or treatment with astrocyte-conditioned medium (ACM) increased the number of inhibitory presynaptic terminals, the frequency of miniature IPSCs, and the number and synaptic localization of GABA(A) receptor (GABA(A)R) clusters during the first 10 d in vitro. We asked whether neurotrophins, which are potent modulators of inhibitory synaptic structure and function, mediate the effects of astrocytes on inhibitory synapses. ACM from BDNF- or tyrosine receptor kinase B (TrkB)-deficient astrocytes increased inhibitory presynaptic terminals and postsynaptic GABA(A)R clusters in wild-type neurons, suggesting that BDNF and TrkB expression in astrocytes is not required for these effects. In contrast, although the increase in the number of inhibitory presynaptic terminals persisted, no increase was observed in postsynaptic GABA(A)R clusters after ACM treatment of hippocampal neurons lacking BDNF or TrkB. These results suggest that neurons, not astrocytes, are the relevant source of BDNF and are the site of TrkB activation required for postsynaptic GABA(A)R modulation. These data also suggest that astrocytes may modulate postsynaptic development indirectly by stimulating Trk signaling between neurons. Together, these data show that astrocytes modulate inhibitory synapse formation via distinct presynaptic and postsynaptic mechanisms.