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Objective: To study the different factors affecting platelet production post transplantation of hematopoietic stem cells (HSCs) isolated from different sources in order to explore novel options for treating platelet depletion following HSCs transplantation. Methods: HSCs and their downstream derivatives including myeloid and lymphoid cells (i.e., collective of mononuclear cells (MNCs)), were isolated from E14.5 fetal liver (FL) and bone marrow (BM) of 8-week-old mice by Ficoll separation technique. These cells were subsequently transplanted into the tibia bone marrow cavity of recipient mice post lethal myeloablative treatment in order to construct the FL-MNCs and BM-MNCs transplantation mouse model. Routine blood indices were examined in these recipient mice. The chimeric rate of donor cells in recipient peripheral blood cells were determined by flow cytometry. Different groups of cells involved in platelet reconstruction were analyzed. CD41+megakaryocytes were sorted from fetal liver or bone marrow using magnetic beads, which were then induced to differentiate into platelets in an in vitro assay. Quantitative RT-PCR was used to detect the expression of platelet-related genes in CD41+megakaryocytes from the two sources. Results: Both the FL-MNCs and the BM-MNCs transplantation groups resumed normal hematopoiesis at the 4th week after transplantation, and the blood cells of the recipient mice were largely replaced by the donor cells. Compared with the mice transplanted with BM-MNCs, the platelet level of mice transplanted with FL-MNCs recovered faster and were maintained at a higher level. At week 4, the PLT level of the FL-MNCs group was (1.45±0.37)×1012/L, and of the BM-MNCs group was (1.22±0.24)×1012/L, P<0.05. The FL-MNCs contain a higher proportion of hematopoietic stem cells (Lin-Sca-1+c-Kit+)(7.60%±1.40%) compared to the BM-MNCs (1.10%±0.46%), P<0.01; the proportion of the megakaryocyte progenitor cells (Lin-Sca-1-c-Kit+CD41+CD150+) and mature megakaryocyte cells (CD41+CD42b+), also differ significantly between the FL-MNCs (3.05%±0.22%, 1.60%±0.06%, respectively) and the BM-MNCs (0.15%±0.02%, 0.87%±0.11%, respectively) groups, both P<0.01. In vitro functional studies showed that FL-MNCs-CD41+megakaryocytes could produce proplatelet-like cells more quickly after induction, with proplatelet-like cells formation on day 3 and significant platelet-like particle formation on day 5, in contrast to bone marrow-derived BM-MNCs-CD41+megakaryocytes that failed to form proplatelet-like cell on day 5. In addition, FL-MNCs-CD41+cells expressed higher levels of platelet-related genes, Mpl (3.25-fold), Fog1 (3-fold), and Gata1 (1.5-fold) (P<0.05). Conclusion: Compared with the BM-MNCs group, the FL-MNCs transplantation group appears to have a more efficient platelet implantation effect in the HSCs transplantation recipient in vivo, as well as a higher platelet differentiation rate in vitro. This might be related to a higher proportion of megakaryocytes and higher expression levels of genes such as Mpl, Fog1, and Gata1 that could be important for platelet formation in FL-MNCs-CD41+cells. Further exploration of the specific functions of these genes and the characteristics of the different proportions of the donor cells will provide valuable clues for the future treatment of platelets reconstitution after HSCs transplantation clinically.
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Plaquetas , Megacariocitos , Animales , Células de la Médula Ósea , Análisis Factorial , Hematopoyesis , Humanos , Hígado , Megacariocitos/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Objective: To explore the effect and mechanism of astaxanthin on acute kidney injury in rats with full-thickness burns. Methods: Forty-eight male Sprague Dawley rats of 8 to 10 weeks were divided into sham injury group, simple burn group, burn+ vehicle group, burn+ low-dose astaxanthin group, burn+ medium-dose astaxanthin group, and burn+ high-dose astaxanthin group according to the random number table, with 8 rats in each group. The back skin of rats in sham injury group were immersed in warm water of 20 â for 15 s to simulate burn injury, and the back skin of rats in the other 5 groups were immersed in boiled water of 100 â for 15 s to inflict full-thickness burn of 30% total body surface area. Fluid resuscitation was performed in rats in the 5 groups except of sham injury group immediately and 6 h after injury. At 30 min after injury, the rats in sham injury group and simple burn group were injected with 1 mL/kg normal saline via tail vein, rats in burn+ vehicle group were injected with 1 mL/kg astaxanthin solvent via tail vein, and rats in burn+ low-dose astaxanthin group, burn+ medium-dose astaxanthin group, and burn+ high-dose astaxanthin group were respectively injected with 5, 10, 20 mg/kg astaxanthin solution of 5, 10, 20 mg/mL via tail vein. The renal tissue was collected at post injury hour (PIH) 48, and hematoxylin eosin staining was used for histopathological observation and renal tubular injury score. At PIH 48, the venous blood was collected for detecting serum creatinine level through blood biochemical analyzer, and blood urea nitrogen (BUN) level was detected by enzyme-linked immunosorbent assay. The renal tissue was collected to detect the mRNA expressions of myeloperoxidase (MPO), interleukin-1ß (IL-1ß), and IL-6 by real-time fluorescent quantitative reverse transcription polymerase chain reaction method, and the protein expressions of Toll like receptor 4 (TLR4), phosphorylated nuclear factor kappa B (p-NF-кB) p65, and heme oxygenase 1 (HO-1) were detected by Western blotting. Besides, the expression of HO-1 in renal tissue was detected by immunofluorescence method. Data were statistically analyzed with Kruskal-Wallis H test, Dunn-Sidák correction, one-way analysis of variance, and Bonferroni method. Results: (1) At PIH 48, there were no inflammatory cell infiltrating and degeneration or necrosis of cells in renal tissue of rats in sham injury group, and the structure of renal tubules was intact. The renal tubules of burn rats in each group showed injury manifestation of separation between epithelial cell and basement membrane, and vacuole cells and lysate protein aggregation. The injury degree of renal tissue of rats in burn+ high-dose astaxanthin group was obviously decreased compared with that in simple burn group. (2) At PIH 48, compared with that of sham injury group, the renal tubular damage scores of rats in simple burn group, burn+ vehicle group, burn+ low-dose astaxanthin group, and burn+ medium-dose astaxanthin group were significantly increased (P<0.05 or P<0.01). Compared with those of simple burn group and burn+ vehicle group, the renal tubular damage scores of rats in burn+ medium-dose astaxanthin group and burn+ high-dose astaxanthin group were significantly decreased (P<0.05 or P<0.01). Compared with that of burn+ low-dose astaxanthin group, the renal tubular damage score of rats in burn+ high-dose astaxanthin group was significantly decreased (P<0.01). (3) At PIH 48, the level of serum creatinine of rats in sham injury group was (2.42±0.06) mg/L, which was significantly lower than (6.11±0.11), (6.48±0.08), (5.79±0.09), (4.03±0.12) mg/L of simple burn group, burn+ vehicle group, burn+ low-dose astaxanthin group, and burn+ medium-dose astaxanthin group (P<0.05 or P<0.01). The level of BUN of rats was (21.9±1.3) mmol/L in sham injury group, significantly lower than (32.1±7.4) mmol/L of simple burn group and (30.2±4.8) mmol/L of burn+ vehicle group (P<0.05 or P<0.01). At PIH 48, compared with those of simple burn group and burn+ vehicle group, the levels of serum creatinine and BUN of (16.0±2.9) mmol/L in burn+ medium-dose astaxanthin group, serum creatinine of (3.02±0.08) mg/L and BUN of (14.5±2.9) mmol/L in burn+ high-dose astaxanthin group, and serum creatinine of (22.8±5.5) mmol/L of rats in burn+ low-dose astaxanthin group were significantly decreased (P<0.05 or P<0.01). At PIH 48, compared with those of burn+ low-dose astaxanthin group, the levels of serum creatinine and BUN of burn+ high-dose astaxanthin group and serum creatinine of burn+ medium-dose group were obviously decreased (P<0.05 or P< 0.01). (4) At PIH 48, compared with those of sham injury group, the mRNA expressions of MPO, IL-1ß, and IL-6 in renal tissue of rats in simple burn group, burn+ vehicle group, burn+ low-dose astaxanthin group, and burn+ medium dose astaxanthin group, and the mRNA expressions of IL-1ß and IL-6 in renal tissue of rats in burn+ high-dose astaxanthin group were obviously increased (P<0.01). Compared with those of simple burn group and burn+ vehicle group, the mRNA expressions of MPO, IL-1ß, and IL-6 in renal tissue of rats were significantly decreased in burn+ low-dose astaxanthin group, burn+ medium-dose astaxanthin group, and burn+ high-dose astaxanthin group (P<0.01). Compared with those of burn+ low-dose astaxanthin group, the mRNA expressions of MPO, IL-1ß, and IL-6 in renal tissue of rats were significantly decreased in burn+ medium-dose astaxanthin group and burn+ high-dose astaxanthin group (P<0.01). The mRNA expressions of MPO, IL-1ß, and IL-6 in renal tissue of rats in burn+ high-dose astaxanthin group were significantly decreased compared with those of burn+ medium-dose astaxanthin group (P<0.01). (5) At PIH 48 h, compared with those of sham injury group, the protein expressions of TLR4 and p-NF-кB p65 in renal tissue of rats in simple burn group, burn+ vehicle group, burn+ low-dose astaxanthin group, and burn+ high-dose astaxanthin group were obviously increased (P<0.01). Compared with those of simple burn group, the protein expressions of TLR4 and p-NF-кB p65 in renal tissue of rats in burn+ low-dose astaxanthin group, burn+ medium dose astaxanthin group, and burn+ high-dose astaxanthin group were significantly decreased (P<0.01). (6) The results of Western blotting combined with immunofluorescence method showed that compared with that of sham injury group, the protein expression of HO-1 in renal tissue of rats in burn+ vehicle group, burn+ low-dose astaxanthin group, burn+ medium-dose astaxanthin group, and burn+ high-dose astaxanthin group were significantly increased at PIH 48 (P<0.01), and the protein expression of HO-1 in renal tissue of rats in burn+ medium-dose astaxanthin group and burn+ high-dose astaxanthin group was significantly increased compared with that of simple burn group (P<0.01). Conclusions: Astaxanthin can attenuate the structural damage and functional decline of renal tissue and regulate the release of injury-related inflammatory factors, thus to protect the rats from acute kidney injury after burn. The HO-1/TLR4/NF-кB signaling pathway is the main regulatory mechanism of astaxanthin to achieve anti-inflammation-based renoprotection.
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Lesión Renal Aguda , Quemaduras , Traumatismos de los Tejidos Blandos , Animales , Masculino , Ratas , Ratas Sprague-Dawley , XantófilasRESUMEN
Wound healing is a complex and sequential biological process involving various cells and factors under body's regulation. Appropriate interventions play positive roles in promoting effective wound healing and improving healing quality. In the clinical practice, there are many new instruments, dressings, and drugs developed for wound care, including antibacterial dressings, wet dressings, precise debridement, negative pressure wound therapy, cytokines/growth factors, and dermal substitutes, which provide revolutionary means for wound treatment. This article summarizes the effective or mature methods in wound care, providing theoretical and practical basis for choosing appropriate treatment methods in different stages of wound.
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Vendajes , Quemaduras/terapia , Terapia de Presión Negativa para Heridas , Cicatrización de Heridas , Apósitos Biológicos , Desbridamiento , Humanos , Piel Artificial , Resultado del TratamientoRESUMEN
There are many factors that may affect the microenvironment of acute and chronic wounds. This article reviews the relationship between temperature factor in the external microenvironment of wound surface and wound healing. The temperature changes in different types and stages of wounds are closely related to the wound healing status. Therefore, wound temperature monitoring provides timely, reliable, and non-invasive method in the evaluation of wound status. As low temperature affects the physiological state of wound, relieving the low temperature state and maintaining normal temperature of the microenvironment of wound can promote wound healing. Further research is needed on the wound repair related effector cell proliferation and the mechanism of regulatory function to determine the optimal constant temperature and heat treatment duration needed for wound healing.
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Quemaduras/terapia , Temperatura , Cicatrización de Heridas/fisiología , Heridas y Lesiones/terapia , Proliferación CelularRESUMEN
Postoperative glaucoma is one of the most common complications after congenital cataract surgery and an important cause of unsatisfactory long-term visual acuity rehabilitation. Recently, the diagnostic criteria of glaucoma after congenital cataract surgery have been gradually complemented; the measurement of intraocular pressure is improved and the follow-up is standardized. The risk factors of glaucoma after congenital cataract surgery include age at surgery, ocular abnormalities and central corneal thickness, some of which still remain controversial. The main treatment of postoperative glaucoma is surgery, while some patients need adjuvant medications in addition to surgery. (Chin J Ophthalmol, 2017, 53: 863-867).
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Extracción de Catarata , Catarata , Glaucoma , Catarata/congénito , Extracción de Catarata/efectos adversos , Estudios de Seguimiento , Humanos , Presión Intraocular , Complicaciones Posoperatorias , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
In this study, the applicability of calcined starfish (SF) and iron-coated SF (ICSF) as potential adsorbents for the treatment of wastewater containing heavy metal ions was evaluated. ICSF was prepared by mixing FeCl(3) solution previously adjusted to pH 7 approximately 9 with SF at 105 degrees C. From the dissolution test at pH 2, ICSF showed strong acid-proof properties. In the batch adsorption, Cu(II) adsorption onto ICSF was completed within 150 minutes, while 47% Cu(II) was removed with SF alone. This result clearly suggests that the coated Fe(III) serves additional adsorption sites, resulting in the enhanced removal of heavy metal ions. The removed fraction of both Cu(II) and Pb(II) increased with increasing solution pH and nearly complete removals of Pb(II) and Cu(II) were observed at around pH 6 and 8, respectively. From the adsorption isotherm of Cu(II) onto SF and ICSF at pH 3.0, the removed amount of Cu(II) by ICSF was greater than that by SF over the entire concentration range studied. In the column test, the breakthrough of Cu(II) in the ICSF column was greatly retarded compared to that in the SF column. Based on the drinking water regulations for Cu(II), SF and ICSF were able to remove 3400 and 8600 mg/kg of Cu(II) from the wastewater, respectively.
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Hierro/química , Metales Pesados/aislamiento & purificación , Estrellas de Mar/química , Contaminantes Químicos del Agua/aislamiento & purificación , Adsorción , Animales , Metales Pesados/química , Purificación del Agua/métodosRESUMEN
The aim of this study was to explore the applicability of manganese coated sand (MCS) in the presence and absence of sodium hypochlorite for the removal of Mn(II) (2 mg/L) from aqueous solutions. Sand itself is widely used as a filter media for the treatment of wastewaters and it was reported that during the treatment, Mn(II), which is present in the wastewater, is to be deposited on the surface of sand in the form of manganese dioxide. The present investigation dealt with various MCS samples, prepared in the laboratory by various doses of Mn(II) (i.e. from 0.05 to 0.2 mol/L) and the samples were obtained from the pilot plant and naturally coated in the water treatment plant for the removal of Mn(II) in the batch and column studies. Moreover, it was realised that the role of hypochlorite is multifunctional as it not only enhances the uptake of Mn(II) on the surface of MCS through oxidation of Mn(II) into Mn(IV) and hence the formation of manganese dioxide, but it was also supposed to disinfect the bacteria or harmful pathogens from the waste/surface waters. The results obtained clearly inferred that various MCS samples used for the removal of Mn(II) from aqueous solutions showed comparable removal efficiency. However, the presence of sodium hypochlorite greatly enhanced the removal of Mn(II) as more than 80% Mn(II) was removed in the presence of sodium hypochlorite at around pH 6.5. Similarly, while comparing the column data it was again noted that the breakthrough points occurred after the 4,100 and 6,500 bed volumes, respectively, in the absence and in the presence of sodium hypochlorite (2 mg/L).
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Manganeso/química , Manganeso/aislamiento & purificación , Dióxido de Silicio/química , Contaminantes Químicos del Agua/química , Contaminantes Químicos del Agua/aislamiento & purificación , Cloro/química , Concentración de Iones de Hidrógeno , SolucionesRESUMEN
Histone deacetylase (HDAC) inhibitors are currently being tested as anticancer agents in clinical trials. Chromatin remodeling, such as through histone acetylation, is a fundamental phenomenon in eukaryotic cell biology, bearing implications to numerous physiological and pathological phenomena. Here, we discuss recent data from our own laboratory and those of others demonstrating antifibrotic and renoprotective effect of HDAC inhibitors in diabetic kidneys, and the possible mechanisms including the role of reactive oxygen species. HDAC inhibitors may prove to be a novel class of multitarget agents in the treatment of diabetic nephropathy.
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Nefropatías Diabéticas/prevención & control , Inhibidores Enzimáticos/uso terapéutico , Inhibidores de Histona Desacetilasas , Acetilación , Animales , Antineoplásicos/uso terapéutico , Nefropatías Diabéticas/tratamiento farmacológico , Histona Desacetilasas/fisiología , Histonas/metabolismo , Humanos , Isoenzimas/fisiología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Transcripción Genética/fisiologíaRESUMEN
There is increasing evidence that reactive oxygen species (ROS) play a major role in the development of diabetic complications. Oxidative stress is increased in diabetes and in chronic kidney disease (CKD). High glucose upregulates transforming growth factor-beta1 (TGF-beta1) and angiotensin II (Ang II) in renal cells and high glucose, TGF-beta1, and Ang II all generate and signal through ROS. ROS mediate high glucose-induced activation of protein kinase C and nuclear factor-kappaB in renal cells. Intensive glycemic control and inhibition of Ang II delay the onset and progression of diabetic nephropathy, in part, through antioxidant activity. Conventional and catalytic antioxidants were shown to prevent or delay the onset of diabetic nephropathy. Transketolase activators and poly (ADP-ribose) polymerase inhibitors were shown to block major biochemical pathways of hyperglycemic damage. Combination of strategies to prevent overproduction of ROS, to increase the removal of preformed ROS, and to block ROS-induced activation of biochemical pathways leading to cellular damage may prove to the effective in preventing the development and progression of CKD in diabetes.
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Nefropatías Diabéticas/prevención & control , Nefropatías Diabéticas/fisiopatología , Especies Reactivas de Oxígeno/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Nefropatías Diabéticas/tratamiento farmacológico , Progresión de la Enfermedad , Humanos , Fallo Renal Crónico , Estrés Oxidativo/fisiologíaRESUMEN
Hyperglycemia-induced oxidative stress and protein kinase C (PKC) activation are implicated in the development and progression of diabetic nephropathy. Although PKC activation under hyperglycemia largely is related to an increase in de novo synthesis of diacylglycerol (DAG), activation of PKC can be regulated sensitively by oxidative stress. We investigated the expression and translocation of PKC isoforms in streptozotocin (STZ)-induced diabetic rat glomeruli and tubules and the effect of an antioxidant taurine. Experimental diabetes was induced by intravenous injection of 50 mg/kg of STZ. Two days after STZ, diabetic rats were assigned to one of two groups: untreated or treated with taurine 1% in drinking water. Four weeks after STZ, PKC isoforms were measured by Western blot analysis in the isolated glomeruli and tubules. DAG-dependent PKC isoforms PKC-alpha, PKC-betaI, PKC-betaII, PKC-delta, and PKC-epsilon and DAG-independent PKC-zeta all were detected in control rat glomeruli and tubules. Streptozotocin increased plasma glucose from 167 +/- 11 mg/dL to 575 +/- 35 mg/dL (n = 9, P < 0.01) and lipid peroxidation from 1.9 +/- 0.2 nmol/mL to 4.2 +/- 0.6 nmol/mL (P < 0.05) and induced proteinuria. In diabetic glomeruli, membrane-associated PKC-delta and PKC-epsilon content increased 47% and 57% above control, and membrane PKC-betaI content decreased to 67% of control. The membrane-associated PKC-alpha, PKC-betaII, and PKC-zeta content were not influenced. Total PKC-delta (163%) and PKC-epsilon (157%) increased significantly in diabetic tubules. Taurine prevented proteinuria and effectively inhibited alterations in PKC-delta and PKC-epsilon of diabetic glomeruli and tubules at dose-inhibiting lipid peroxidation but not hyperglycemia. These data suggest that PKC-delta and PKC-epsilon are sensitively activated by hyperglycemia-induced oxidative stress in diabetic rat kidney.
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Diabetes Mellitus/fisiopatología , Isoenzimas/metabolismo , Riñón/fisiopatología , Estrés Oxidativo , Proteína Quinasa C/metabolismo , Animales , Diabetes Mellitus/inducido químicamente , Progresión de la Enfermedad , Activación Enzimática , Hiperglucemia/fisiopatología , Túbulos Renales/enzimología , Masculino , Proteína Quinasa C-delta , Proteína Quinasa C-epsilon , Ratas , Ratas Sprague-Dawley , EstreptozocinaRESUMEN
BACKGROUND: Progressive peritoneal fibrosis, membrane hyperpermeability, and ultrafiltration failure have been observed in long-term peritoneal dialysis (PD) using glucose as an osmotic agent. High glucose activates protein kinase C (PKC), which is one important signal pathway in the activation of transforming growth factor-beta 1 (TGF-beta 1) and fibronectin (FN). To gain a better understanding of mechanisms involved in peritoneal fibrosis, we examined the effects of high glucose on human peritoneal mesothelial cell (HPMC) TGF-beta 1 and FN mRNA expression and protein synthesis and determined the involvement of PKC in the high glucose-induced HPMC activation. METHODS: Synchronized confluent HPMC were incubated with different concentrations of glucose with and without inhibition of PKC. PKC activity and diacylglycerol (DAG) levels were measured. The expression of TGF-beta 1 and FN mRNAs by HPMC was measured by Northern blot analysis. TGF-beta 1 protein was measured by enzyme-linked immunosorbent assay (ELISA) and mink lung epithelial cell growth inhibition assay. FN protein was measured by Western blot analysis and ELISA. RESULTS: PKC activity and DAG levels in HPMC cultured under 50 mmol/L (high) glucose increased 2.3- and 2.0-fold, respectively, that of 5.6 mmol/L (control) glucose at 24 hours and this was sustained up to 72 hours. The expression of TGF-beta 1 and FN mRNA by HPMC cultured under high glucose increased 1.6- and 1.7-fold, respectively, that of control values at 24 hours. TGF-beta bioactivity as well as protein content in heat-activated conditioned media from high glucose was significantly higher than that of control values at 24 and 48 hours. FN protein also increased in response to high glucose, as measured by Western blot analysis and ELISA. PKC activator phorbol 12-myristate 13-acetate (PMA) induced 2.2- and 1.4-fold increase in TGF-beta 1 and FN mRNA expression, respectively. Depletion of PKC and calphostin C, a PKC inhibitor, effectively prevented both PMA and high glucose-induced, but not constitutive, expression of TGF-beta 1 and FN. CONCLUSION: The present data demonstrate that high glucose up-regulates TGF-beta 1 and FN synthesis by HPMC, and that this high glucose-induced up-regulation is largely mediated by PKC. These results suggest that activation of PKC by high glucose in conventional PD solutions may constitute an important signal for activation of HPMC, leading to progressive accumulation of extracellular matrix and eventual peritoneal fibrosis.
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Fibronectinas/biosíntesis , Glucosa/administración & dosificación , Peritoneo/metabolismo , Proteína Quinasa C/metabolismo , Factor de Crecimiento Transformador beta/biosíntesis , Células Cultivadas , Diglicéridos/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/fisiología , Células Epiteliales/metabolismo , Fibronectinas/genética , Glucosa/farmacología , Humanos , Peritoneo/citología , Proteína Quinasa C/fisiología , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta1 , Regulación hacia ArribaRESUMEN
OBJECTIVE: To investigate the biocompatibility of "new" peritoneal dialysis (PD) solutions with bicarbonate/lactate buffer, non glucose osmotic agents (icodextrin or amino acids), neutral pH, and low levels of glucose degradation products (GDPs). DESIGN: Using M199 culture medium as a control, we compared conventional and new PD solutions with respect to their effects on the viability of human peritoneal mesothelial cells (HPMCs) [using lactate dehydrogenase (LDH) release], on DNA damage in HPMCs [using single-cell gel electrophoresis (Comet assay)], and on HPMC proliferation (using [3H]-thymidine incorporation). The experiments were performed after cell growth was synchronized by incubation with serum-free media for 24 hours. The PD solutions tested included commercial 1.5% glucose and 4.25% glucose solutions with 40 mmol/L lactate (D 1.5 and D 4.25, respectively), 7.5% icodextrin (E), 1.1% amino acid (N), 1.5% glucose solution in a triple-chambered bag (Bio 1.5), 1.5% glucose solution in a dual-chambered bag with neutral pH (Bal 1.5), and 1.5% glucose and 4.25% glucose solution containing 25 mmol/L bicarbonate and 15 mmol/L lactate (P 1.5 and P 4.25, respectively). RESULTS: When HPMCs were continuously exposed to undiluted PD solutions, D 1.5, D 4.25, P 4.25, and E increased LDH release by more than 60% at 24 hours. All PD solutions tested increased LDH release by more than 75% at 96 hours. With 2-fold diluted PD solutions, only D 4.25 significantly increased LDH release at 96 hours, though not at 24 hours. When cells were exposed to undiluted PD solutions for 60 min and allowed to recover in M199 for up to 96 hours, LDH release was significantly higher at 24-96 hours in E (55%-69%) and D 1.5 (48%-72%) as compared with control [M199 (18%)]. Release of LDH was significantly lower with PD solutions containing lower levels of GDPs than those in D 1.5, suggesting that GDPs may have a role in cell viability. The D solutions (D 1.5 and D 4.25) and E solution also induced significant DNA damage. Both LDH release and DNA damage by D and E were significantly attenuated by adjusting the solution pH to 7.4, suggesting that low pH may be implicated in PD solution-induced DNA damage and cell death. When diluted 2-fold, D 1.5, D 4.25, and P 4.25 decreased [3H]-thymidine incorporation to 43%, 34%, and 41% of control, respectively, at 24 hours and to 45%, 26%, and 35% of control, respectively, at 96 hours. When cells were exposed to undiluted PD solutions for 5 minutes and allowed to recover in M199 for up to 96 hours, D1.5 and P 4.25--but not D 4.25--significantly inhibited cell proliferation at 24 hours. This effect was sustained up to 96 hours. CONCLUSIONS: The present in vitro data demonstrate that PD solutions with low pH, or high levels of GDPs, or both, promote HPMC death and DNA damage, and that PD solutions with high osmolality inhibit cell proliferation. Solutions with neutral pH, amino acids, and "low GDPs" appear to be more biocompatible than conventional PD solutions. These results require confirmation in in vivo animal and clinical studies.
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Soluciones para Diálisis/farmacología , Células Epiteliales/efectos de los fármacos , Diálisis Peritoneal , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ensayo Cometa , Medios de Cultivo , ADN/efectos de los fármacos , Soluciones para Diálisis/química , Soluciones para Diálisis/toxicidad , Humanos , Concentración de Iones de Hidrógeno , L-Lactato Deshidrogenasa/metabolismo , Cavidad Peritoneal/citologíaRESUMEN
Oxidative stress occurs in diabetic patients and experimental models of diabetes. We examined whether two antioxidants, melatonin and taurine, can ameliorate diabetic nephropathy. Enhanced expression of glomerular TGF-beta1 and fibronectin mRNAs and proteinuria were employed as indices of diabetic nephropathy. Experimental diabetes was induced by intravenous injection of streptozotocin 50 mg/kg. Two days after streptozotocin, diabetic rats were assigned to one of the following groups: i) untreated; ii) melatonin supplement by 0.02% in drinking water; or iii) taurine supplement by 1% in drinking water. Four weeks after streptozotocin, diabetic rats (n = 6: plasma glucose 516+/-12 mg/dl) exhibited 6.1 fold increase in urinary protein excretion, 1.4 fold increase in glomerular TGF-beta1 mRNA, 1.7 fold increase in glomerular fibronectin mRNA, 2.2 fold increase in plasma lipid peroxides (LPO), and 44 fold increase in urinary LPO excretion above the values in control rats (n = 6: plasma glucose 188+/-14 mg/dl). Chronic administration of melatonin (n = 6) and taurine (n = 6) prevented increases in glomerular TGF-beta1 and fibronectin mRNAs and proteinuria without having effect on blood glucose. Both treatments reduced lipid peroxidation by nearly 50%. The present data demonstrate beneficial effects of melatonin and taurine on early changes in diabetic kidney and suggest that diabetic nephropathy associated with hyperglycemia is largely mediated by oxidative stress.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Nefropatías Diabéticas/prevención & control , Glomérulos Renales/efectos de los fármacos , Melatonina/farmacología , Taurina/farmacología , Animales , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Nefropatías Diabéticas/fisiopatología , Diuresis/efectos de los fármacos , Fibronectinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Glomérulos Renales/patología , Glomérulos Renales/fisiopatología , Peroxidación de Lípido , Masculino , Estrés Oxidativo , Proteinuria , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Transcripción Genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
BACKGROUND: Transforming growth factor (TGF)-beta is recognized as the final common mediator of the principal lesions of diabetic nephropathy such as renal hypertrophy and mesangial expansion. To gain better understanding of the temporal relationships between high glucose (HG) and mesangial cell (MC) TGF-beta 1 synthesis and between TGF-beta 1 and extracellular matrix (ECM) synthesis, the present study examined early and sequential effects of HG on TGF-beta 1 and fibronectin (FN) mRNA expression and protein synthesis. METHODS: Confluent primary rat MC was stimulated with 5.6 (control) or 30 (high) mM glucose after synchronizing the growth by incubation with serum-free media for 48 hours. RESULTS: Mesangial cell TGF-beta 1 mRNA expression increased significantly in six hours and continued to increase until 48 hours in response to HG. The level of TGF-beta 1 mRNA was 1.5-fold higher than that of control glucose at six hours and 1.8-fold at 48 hours. TGF-beta activity in heat-activated conditioned media under HG increased 1.5- and 1.6-fold at 24 and 48 hours, respectively, compared to control glucose. FN mRNA increased significantly at 24 and 48 hours and 1.4-fold that of control glucose at both time points. FN protein also increased 1.5-fold that of control glucose at 48 hours. Anti-TGF-beta antibody completely abolished HG-induced FN synthesis. CONCLUSIONS: The present finding demonstrate that HG stimulates TGF-beta 1 very early and prior to FN production and that HG-induced FN production is mediated by TGF-beta. This finding is consistent with the view that TGF-beta mediates increased ECM accumulation by MC under high glucose conditions.