RESUMEN
Dual immunoglobulin domain-containing cell adhesion molecule (DICAM) is a type I transmembrane protein that presents in various cells including renal tubular cells. This study evaluated the expression and protective role of DICAM in renal tubular cell injury. HK-2 cells were incubated and treated with lipopolysaccharide (LPS, 30 µg/mL) or hydrogen peroxide (H2O2, 100 µM) for 24 h. To investigate the effect of the gene silencing of DICAM, small interfering RNA of DICAM was used. Additionally, to explain its role in cellular response to injury, DICAM was overexpressed using an adenoviral vector. DICAM protein expression levels significantly increased following treatment with LPS or H2O2 in HK-2 cells. In response to oxidative stress, DICAM showed an earlier increase (2-4 h following treatment) than neutrophil gelatinase-associated lipocalin (NGAL) (24 h following treatment). DICAM gene silencing increased the protein expression of inflammation-related markers, including IL-1ß, TNF-α, NOX4, integrin ß1, and integrin ß3, in H2O2-induced HK-2 cell injury. Likewise, in the LPS-induced HK-2 cell injury, DICAM knockdown led to a decrease in occludin levels and an increase in integrin ß3, IL-1ß, and IL-6 levels. Furthermore, DICAM overexpression followed by LPS-induced HK-2 cell injury resulted in an increase in occludin levels and a decrease in integrin ß1, integrin ß3, TNF-α, IL-1ß, and IL-6 levels, suggesting an alleviating effect on inflammatory responses. DICAM was elevated in the early stage of regular tubular cell injury and may protect against renal tubular injury through its anti-inflammatory properties. DICAM has a potential as an early diagnostic marker and therapeutic target for renal cell injury.
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Recently, the role of kidney pericytes in kidney fibrosis has been investigated. This study aims to evaluate the effect of paricalcitol on hypoxia-induced and TGF-ß1-induced injury in kidney pericytes. The primary cultured pericytes were pretreated with paricalcitol (20 ng/mL) for 90 min before inducing injury, and then they were exposed to TGF-ß1 (5 ng/mL) or hypoxia (1% O2 and 5% CO2). TGF-ß1 increased α-SMA and other fibrosis markers but reduced PDGFRß expression in pericytes, whereas paricalcitol reversed the changes. Paricalcitol inhibited the TGF-ß1-induced cell migration of pericytes. Hypoxia increased TGF-ß1, α-SMA and other fibrosis markers but reduced PDGFRß expression in pericyte, whereas paricalcitol reversed them. Hypoxia activated the HIF-1α and downstream molecules including prolyl hydroxylase 3 and glucose transporter-1, whereas paricalcitol attenuated the activation of the HIF-1α-dependent molecules and TGF-ß1/Smad signaling pathways in hypoxic pericytes. The gene silencing of HIF-1α vanished the hypoxia-induced TGF-ß1, α-SMA upregulation, and PDGFRß downregulation. The effect of paricalcitol on the HIF-1α-dependent changes of fibrosis markers was not significant after the gene silencing of HIF-1α. In addition, hypoxia aggravated the oxidative stress in pericytes, whereas paricalcitol reversed the oxidative stress by increasing the antioxidant enzymes in an HIF-1α-independent manner. In conclusion, paricalcitol improved the phenotype changes of pericyte to myofibroblast in TGF-ß1-stimulated pericytes. In addition, paricalcitol improved the expression of fibrosis markers in hypoxia-exposed pericytes both in an HIF-1α-dependent and independent manner.
Asunto(s)
Ergocalciferoles/farmacología , Hipoxia/metabolismo , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Sustancias Protectoras/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Células Cultivadas , Fibrosis , Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Ratones , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Estrés Oxidativo , Pericitos/patología , Fosforilación , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismoRESUMEN
In the present study, we investigated the effects of xanthine oxidase (XO) inhibition on cholesterol-induced renal dysfunction in chronic kidney disease (CKD) mice, and in low-density lipoprotein (LDL)-treated human kidney proximal tubule epithelial (HK-2) cells. ApoE knockout (KO) mice underwent uninephrectomy to induce CKD, and were fed a normal diet or high-cholesterol (HC) diet along with the XO inhibitor topiroxostat (1 mg/kg/day). HK-2 cells were treated with LDL (200 µg/mL) and topiroxostat (5 µM) or small interfering RNA against xanthine dehydrogenase (siXDH; 20 nM). In uninephrectomized ApoE KO mice, the HC diet increased cholesterol accumulation, oxidative stress, XO activity, and kidney damage, while topiroxostat attenuated the hypercholesterolemia-associated renal dysfunction. The HC diet induced cholesterol accumulation by regulating the expressions of genes involved in cholesterol efflux (Nr1h3 and Abca1) and synthesis (Srebf2 and Hmgcr), which was reversed by topiroxostat. Topiroxostat suppressed the expressions of genes related to hypercholesterolemia-associated inflammation and fibrosis in the unilateral kidney. LDL stimulation evoked changes in the cholesterol metabolism, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and NF-κB pathways in HK-2 cells, which were mitigated by XO inhibition with topiroxostat or siXDH. These findings suggest that XO inhibition exerts renoprotective effects against hypercholesterolemia-associated kidney injury. XO could be a novel therapeutic target for hypercholesterolemia-associated kidney injury in uninephrectomized patients.
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Hipercolesterolemia/complicaciones , Hipercolesterolemia/metabolismo , Insuficiencia Renal Crónica/etiología , Insuficiencia Renal Crónica/metabolismo , Xantina Oxidasa/metabolismo , Colesterol/metabolismo , Progresión de la Enfermedad , Susceptibilidad a Enfermedades , Fibrosis , Humanos , Metabolismo de los Lípidos , Lipoproteínas LDL/metabolismo , Estrés Oxidativo , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/patología , Transducción de Señal , Xantina Oxidasa/genéticaRESUMEN
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.
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Epithelial-to-mesenchymal transition (EMT) is one of the main causes of peritoneal fibrosis. However, the pathophysiological mechanisms of EMT, specifically its relationship with autophagy, are still unknown. This study aimed to evaluate the role of autophagy in transforming growth factor-beta 1 (TGF-ß1)-induced EMT in human peritoneal mesothelial cells (HPMCs). Primary cultured HPMCs were treated with TGF-ß1 (2 and 5 ng/mL) and changes in autophagy markers and the relationship between autophagy and EMT were evaluated. We also identified changes in EMT- and autophagy-related signaling pathways after autophagy and NADPH oxidase 4 (NOX4) inhibition. TGF-ß1 increased the generation of NOX4 and reactive oxygen species (ROS) in HPMCs, resulting in mitochondrial damage. Treatment with GKT137831 (20 µM), a NOX1/4 inhibitor, reduced ROS in the mitochondria of HPMC cells and reduced TGF-ß1-induced mitochondrial damage. Additionally, the indirect inhibition of autophagy by GKT137831 (20 µM) downregulated TGF-ß1-induced EMT, whereas direct inhibition of autophagy using 3-methyladenine (3-MA) (2 mM) or autophagy-related gene 5 (ATG5) gene silencing decreased the TGF-ß1-induced EMT in HPMCs. The suppressor of mothers against decapentaplegic 2/3 (Smad2/3), autophagy-related phosphoinositide 3-kinase (PI3K) class III, and protein kinase B (Akt) pathways, and mitogen-activated protein kinase (MAPK) signaling pathways, such as extracellular signal-regulated kinase (ERK) and P38, were involved in TGF-ß1-induced EMT. Autophagy and NOX4 inhibition suppressed the activation of these signaling pathways. Direct inhibition of autophagy and its indirect inhibition through the reduction of mitochondrial damage by upstream NOX4 inhibition reduced EMT in HPMCs. These results suggest that autophagy could serve as a therapeutic target for the prevention of peritoneal fibrosis in patients undergoing peritoneal dialysis.
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Autofagia , Células Epiteliales , Transición Epitelial-Mesenquimal , NADPH Oxidasa 4 , Estrés Oxidativo , Especies Reactivas de Oxígeno , Transducción de Señal , Factor de Crecimiento Transformador beta1 , Humanos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Autofagia/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , NADPH Oxidasa 4/metabolismo , NADPH Oxidasa 4/genética , Transducción de Señal/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Peritoneo/patología , Pirazolonas , PiridonasRESUMEN
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.
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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: It has been demonstrated that phosphate uptake through the type III sodium-dependent phosphate co-transporter, Pit-1, induced apoptosis of aortic vascular smooth muscle cells and endothelial cells in vitro. However, the apoptotic effects of high phosphate (HP) level in human peritoneal mesothelial cells (HPMCs) are not known. METHODS: To examine whether Pit-1 is expressed in HPMCs, we checked the Western blot assay of immunoreactive Pit-1 and the transcription of Pit-1 by reverse transcriptase PCR. We treated several different phosphate concentrations (1-4 mM) and calcium concentrations (1.8 and 2.8 mM) on HPMCs to assess the effects of concentration. MTT, TUNEL assays, and flow cytometry analysis using Annexin V and propidium iodide were performed to identify cell death and apoptosis. Bax and Bcl-2 by Western blot and caspase-3 activity were evaluated by colorimetric assay. In addition, phosphonoformic acid (PFA) and pan-caspase inhibitor, Z-VAD-FMK, were given to prevent phosphate-induced apoptosis. RESULTS: Pit-1 expression on HPMCs was demonstrated. Apoptosis in HPMCs significantly increased with a high concentration of phosphate in a dose- and time-dependent manner, and was enhanced in the presence of 2.8 mM calcium. HP concentrations significantly decreased the anti-apoptotic Bcl-2/Bax ratio and increased caspase-3 activity. The treatment with PFA and Z-VAD-FMK prevented cell death by HP. CONCLUSION: Phosphate uptake through Pit-1 induces apoptosis in HPMCs by a caspase-related mechanism.
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Apoptosis/fisiología , Caspasa 3/metabolismo , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismo , Anexina A5/metabolismo , Calcio/metabolismo , Supervivencia Celular , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Peritoneo/citología , Peritoneo/metabolismo , Propidio/metabolismo , Transducción de Señal , Proteína X Asociada a bcl-2/metabolismoRESUMEN
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.
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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
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
BACKGROUND: The association between peritoneal solute transport rates (PSTRs) and inflammatory markers in patients on peritoneal dialysis (PD) is still under investigation. We aimed to elucidate their relationship during the first year on PD. METHODS: We performed a prospective observational study with 187 incident PD patients who were treated with either biocompatible solution (BCS) or conventional solution (CS). Peritoneal dialysate effluent (PDE) and blood samples for the markers and the calculation of mass transfer area coefficient of creatinine (MTAC) were performed at 1, 6 and 12 months after commencing PD. RESULTS: Of the 187 enrolled patients, 110 completed a 1-year study protocol. All PDE markers [interleukin-6 (IL-6), transforming growth factor-beta (TGF-beta), TGF-beta-induced gene-h3 (beta ig-h3), vascular endothelial growth factor (VEGF)] except CA125 increased over time, whereas PSTRs, high-sensitivity C-reactive protein (hs-CRP) and serum IL-6 levels did not change. Serum albumin and log PDE appearance rates (ARs) of IL-6, TGF-beta and CA125 predicted MTAC. The Delta value (12-month minus 1-month) of PDE AR of IL-6 was correlated with those of all other PDE markers. Both 12-month IL-6 and Delta IL-6 ARs in PDE were highest in the upper Delta MTAC tertile. PSTRs in the CS group, unlike BCS, had a tendency to increase over time, demonstrating a time-by-group interaction. Solution type and MTAC were not associated with patient and technique survival. CONCLUSIONS: The change in PSTR during the first year of PD is related to PDE IL-6 AR, which may represent intraperitoneal inflammation; however, there does not seem to be a close association between PSTR and the degree of systemic inflammation.
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Inflamación/fisiopatología , Diálisis Peritoneal/efectos adversos , Peritoneo/fisiopatología , Peritonitis/fisiopatología , Adulto , Anciano , Transporte Biológico Activo , Biomarcadores/análisis , Biomarcadores/sangre , Antígeno Ca-125/análisis , Antígeno Ca-125/sangre , Creatinina/análisis , Creatinina/sangre , Citocinas/análisis , Citocinas/sangre , Soluciones para Diálisis , Femenino , Humanos , Mediadores de Inflamación/análisis , Mediadores de Inflamación/sangre , Interleucina-6/análisis , Interleucina-6/sangre , Estimación de Kaplan-Meier , Fallo Renal Crónico/fisiopatología , Fallo Renal Crónico/terapia , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/sangre , Persona de Mediana Edad , Permeabilidad , Estudios Prospectivos , Factor de Crecimiento Transformador beta/análisis , Factor de Crecimiento Transformador beta/sangreRESUMEN
The response to erythropoiesis stimulating agents (ESAs) is affected by inflammation linked to middle molecules in hemodialysis (HD) patients. We evaluated the effect of a medium cut-off (MCO) dialyzer on ESA resistance in maintenance HD patients. Forty-nine patients who underwent high-flux HD were randomly allocated to the MCO or high-flux group. The primary outcome was the changes of erythropoietin resistance index (ERI; U/kg/wk/g/dL) between baseline and 12 weeks. The MCO group showed significant decrease in the ESA dose, weight-adjusted ESA dose, and ERI compared to the high-flux group at 12 weeks (p < 0.05). The generalized estimating equation models revealed significant interactions between groups and time for the ESA dose, weight-adjusted ESA dose, and ERI (p < 0.05). Serum iron and transferrin saturation were higher in the MCO group at 12 weeks (p < 0.05). The MCO group showed a greater reduction in TNF-α and lower serum TNF-α level at 12 weeks compared to the high-flux group (p < 0.05), whereas no differences were found in the reduction ratio of hepcidin and serum levels of erythropoietin, erythroferrone, soluble transferrin receptor and hepcidin between groups. HD with MCO dialyzer improves ESA resistance over time compared to high-flux HD in maintenance HD patients. The MCO dialyzer provides superior removal of the inflammatory cytokine and thus improves iron metabolism in a hepcidin-independent manner.
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Hematínicos/farmacología , Hepcidinas/farmacología , Diálisis Renal/métodos , Anciano , Anemia , Proteína C-Reactiva/metabolismo , Eritropoyesis/efectos de los fármacos , Eritropoyetina/metabolismo , Femenino , Hepcidinas/metabolismo , Humanos , Inflamación , Hierro/metabolismo , Fallo Renal Crónico/sangre , Fallo Renal Crónico/fisiopatología , Masculino , Persona de Mediana Edad , Receptores de Transferrina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Medium cut-off (MCO) dialyzers help remove larger middle molecules associated with symptoms related to the accumulation of uremic retention solutes. We investigated the effect of an MCO dialyzer on the improvement of quality of life (QOL) in maintenance hemodialysis (HD) patients. Forty-nine HD patients with high-flux dialysis were randomly assigned to either an MCO (Theranova 400, Baxter) or a high-flux (FX CorDiax 80 or 60, Fresenius Medical Care) dialyzer and completed the study. QOL was assessed at baseline and after 12 weeks of treatment using the Kidney Disease Quality of Life Short Form-36, and pruritus was assessed using a questionnaire and visual analog scale. The reduction ratios of middle molecules were also evaluated. Laboratory markers, including serum albumin, did not differ between the two groups after 12 weeks. Removals of kappa and lambda free light chains were greater for MCO dialyzer than high-flux dialyzer. The MCO group had higher scores than the high-flux group in the domains of physical functioning and physical role (75.2 ± 20.8 vs. 59.8 ± 30.1, P = 0.042; 61.5 ± 37.6 vs. 39.0 ± 39.6, P = 0.047, respectively), and the MCO group had lower mean scores for morning pruritus distribution and the frequency of scratching during sleep (1.29 ± 0.46 vs. 1.64 ± 0.64, P = 0.034; 0.25 ± 0.53 vs. 1.00 ± 1.47, P = 0.023, respectively). MCO dialyzers may improve patient-reported outcomes, particularly the physical components of QOL and uremic pruritus, in patients with high-flux dialyzers.
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Fallo Renal Crónico/epidemiología , Fallo Renal Crónico/terapia , Calidad de Vida , Diálisis Renal , Anciano , Biomarcadores , Comorbilidad , Femenino , Hemodiafiltración , Humanos , Fallo Renal Crónico/metabolismo , Fallo Renal Crónico/mortalidad , Masculino , Persona de Mediana Edad , Diálisis Renal/efectos adversos , Diálisis Renal/instrumentación , Diálisis Renal/métodos , Resultado del TratamientoRESUMEN
BACKGROUND: The alpha-1 antitrypsin (AAT) protein has an important role in the anti-inflammatory and apoptotic response. AAT inhibits not only serine proteases but also cysteine and aspartic proteases. Apoptosis results from the sequential activation of cysteine proteases of the caspase family. This study aimed to evaluate the effect of AAT on formaldehyde-induced apoptosis of human peritoneal mesothelial cells (HPMCs). METHODS: HPMCs were cultured and treated with formaldehyde (250 µM) to induce apoptosis. In the AAT group, the cultured HPMCs were pretreated with AAT (2 mg/mL) for 1 h before formaldehyde treatment. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays to determine cell viability, and flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays to detect apoptosis. The MTT assays were used to find optimal concentrations of formaldehyde and AAT. We measured caspase-3 activity and used Western blotting to estimate Bcl-2 and Bad expression. RESULTS: Flow cytometry and TUNEL assays revealed that formaldehyde exposure significantly increased apoptosis compared with the control treatment, but pretreatment with AAT significantly inhibited this effect. Compared with the control, caspase-3 activity was significantly increased and the ratio of Bcl-2 to Bad expression significantly decreased following treatment with formaldehyde. However, caspase-3 activity was significantly lower and the Bcl-2 to Bad expression ratio higher in the AAT group than in the formaldehyde-only group. CONCLUSION: AAT inhibits formaldehyde-induced apoptosis of HPMCs via a caspase-mediated pathway. These data support a potential use for AAT as a therapeutic agent for the inhibition of peritoneal cell apoptosis during peritoneal dialysis.
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Apoptosis/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Formaldehído/farmacología , Peritoneo/efectos de los fármacos , Peritoneo/patología , alfa 1-Antitripsina/farmacología , Caspasa 3/metabolismo , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Diálisis Peritoneal , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína Letal Asociada a bcl/metabolismoRESUMEN
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.
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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
As an attempt to find bioactive medicinal herbs exerting anti-asthmatic activity, the effects of an ethanol extract from the parts of Saururus chinensis were evaluated in both in vitro and in vivo. The ethanol extract of S. chinensis (ESC) inhibited generation of the cyclooxygenase-2 (COX-2) dependent phases of prostaglandin D(2) in bone marrow-derived mast cells in a concentration-dependent manner with an IC(50) value of 14.3 microg/ml. ESC also inhibited leukotriene C(4) production with an IC(50) value of 0.3 microg/ml. This demonstrates that ESC has COX-2/5-lipoxygenase dual inhibitory activity. In addition, this compound inhibited degranulation reaction in a dose dependent manner, with an IC(50) value of 1.3 microg/ml. An ovalbumin induced mouse asthmatic animal model was used to determine its in vivo anti-asthmatic activity. The oral administration (50-200 mg/kg) of ESC reduced the number of infiltrated eosinophil in a bronchoalveolar lavage fluid. Furthermore, ESC (100 mg/kg) inhibited the eotaxin and IL-4 mRNA expression levels. These results suggest that the anti-asthmatic activity of S. chinensis might in part occur via the inhibition of eicosanoid generation, degranulation as well as the down regulation of IL-4 and eotaxin mRNA expression.