RESUMEN
Metformin, an AMP-activated protein kinase (AMPK) activator, has been shown in previous studies to reduce kidney fibrosis in different models of experimental chronic kidney disease (CKD). However, in all of these studies, the administration of metformin was initiated before the establishment of renal disease, which is a condition that does not typically occur in clinical settings. The aim of the present study was to investigate whether the administration of metformin could arrest the progression of established renal disease in a well-recognized model of CKD, the subtotal kidney nephrectomy (Nx) model. Adult male Munich-Wistar rats underwent either Nx or sham operations. After the surgery (30 days), Nx rats that had systolic blood pressures of >170 mmHg and albuminuria levels of >40 mg/24 h were randomized to a no-treatment condition or to a treatment condition with metformin (300 mg·kg-1·day-1) for a period of either 60 or 120 days. After 60 days of treatment, we did not observe any differences in kidney disease parameters between Nx metformin-treated and untreated rats. However, after 120 days, Nx rats that had been treated with metformin displayed significant reductions in albuminuria levels and in markers of renal fibrosis. These effects were independent of any other effects on blood pressure or glycemia. In addition, treatment with metformin was also able to activate kidney AMPK and therefore improve mitochondrial biogenesis. It was concluded that metformin can arrest the progression of established kidney disease in the Nx model, likely via the activation of AMPK.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Activadores de Enzimas/farmacología , Riñón/efectos de los fármacos , Metformina/farmacología , Nefrectomía , Insuficiencia Renal Crónica/prevención & control , Albuminuria/etiología , Albuminuria/metabolismo , Albuminuria/prevención & control , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Activación Enzimática , Fibrosis , Hipertensión/etiología , Hipertensión/metabolismo , Hipertensión/prevención & control , Riñón/enzimología , Riñón/patología , Riñón/cirugía , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Biogénesis de Organelos , Ratas Wistar , Insuficiencia Renal Crónica/etiología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Factores de TiempoRESUMEN
Reduction in sirtuin 1 (Sirt-1) is associated with extracellular matrix (ECM) accumulation in the diabetic kidney. Theobromine may reduce kidney ECM accumulation in diabetic rats. In the current study, we aimed to unravel, under diabetic conditions, the mechanism of kidney ECM accumulation induced by a reduction in Sirt-1 and the effect of theobromine in these events. In vitro, we used immortalized human mesangial cells (iHMCs) exposed to high glucose (HG; 30 mM), with or without small interfering RNA for NOX4 and Sirt-1. In vivo, spontaneously hypertensive rats (SHR) were rendered diabetic by means of streptozotocin and studied after 12 wk. The effects of treatment with theobromine were investigated under both conditions. HG leads to a decrease in Sirt-1 activity and NAD(+) levels in iHMCs. Sirt-1 activity could be reestablished by treatment with NAD(+), silencing NOX4, and poly (ADP-ribose) polymerase-1 (PARP-1) blockade, or with theobromine. HG also leads to a low AMP/ATP ratio, acetylation of SMAD3, and increased collagen IV, which is prevented by theobromine. Sirt-1 or AMPK blockade abolished these effects of theobromine. In diabetic SHR, theobromine prevented increases in albuminuria and kidney collagen IV, reduced AMPK, elevated NADPH oxidase activity and PARP-1, and reduced NAD(+) levels and Sirt-1 activity. These results suggest that in diabetes mellitus, Sirt-1 activity is reduced by PARP-1 activation and NAD(+) depletion due to low AMPK, which increases NOX4 expression, leading to ECM accumulation mediated by transforming growth factor (TGF)-ß1 signaling. It is suggested that Sirt-1 activation by theobromine may have therapeutic potential for diabetic nephropathy.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Riñón/metabolismo , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 1/metabolismo , Teobromina/farmacología , Animales , Diabetes Mellitus Experimental/tratamiento farmacológico , Modelos Animales de Enfermedad , Riñón/efectos de los fármacos , Masculino , Estrés Oxidativo/fisiología , Poli(ADP-Ribosa) Polimerasa-1 , Ratas , Ratas Endogámicas SHRRESUMEN
BACKGROUND/AIMS: High phosphate (Pi) levels and extracellular matrix (ECM) accumulation are associated with chronic kidney disease progression. However, how high Pi levels contribute to ECM accumulation in mesangial cells is unknown. The present study investigated the role and mechanism of high Pi levels in ECM accumulation in immortalized human mesangial cells (iHMCs). METHODS: iHMCs were exposed to normal (0.9 mM) or increasing Pi concentrations (2.5 and 5 mM) with or without diferent blockers or activators. NOX4, phosphorylated AMPK (p-AMPK), phosphorylated SMAD3 (p-SMAD3), fibronectin (F/N), collagen IV (C-IV) and alpha-smooth muscle actin (α-SMA) expression was assessed via western blot and immunofluorescence. Lucigenin-enhanced chemiluminescence, and dihydroethidium (DHE) assessed NADPH oxidase activity and superoxide (SO), respectively. RESULTS: In iHMCs, a Pi transporter blocker (PFA) abrogated high Pi-induced AMPK inactivation, increase in NADPH oxidase-induced reactive oxygen species (ROS) levels, NOX4, p-SMAD3, α-SMA and C-IV expression. AMPK activation by AICAR, NOX4 silencing or NADPH oxidase blocker prevented high Pi-induced DHE levels, p-SMAD3, F/N, C-IV and α-SMA expression. CONCLUSION: AMPK inactivation with NOX4-induced ROS formation and transforming growth factor ß-1 (TGFß-1) signaling activation mediates high Pi-induced ECM accumulation in iHMCs. Maneuvers increasing AMPK or reducing NOX4 activity may contribute to renal protection under hyperphosphatemia.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Matriz Extracelular/metabolismo , Células Mesangiales/metabolismo , NADPH Oxidasas/metabolismo , Fosfatos/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Actinas/metabolismo , Células Cultivadas , Colágeno Tipo IV/metabolismo , Fibronectinas/metabolismo , Humanos , NADPH Oxidasa 4 , Fosforilación/fisiología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Proteína smad3/metabolismo , Superóxidos/metabolismoRESUMEN
BACKGROUND/AIMS: In diabetic hypertensive rats, tempol reduces albuminuria by restoring the redox imbalance. Increased formation of reactive oxygen species leading to activation of poly(ADP-ribose) polymerase (PARP)-1 and podocyte loss by apoptosis contribute to albuminuria in diabetes mellitus (DM). In the present study, we investigated the hypothesis that in DM tempol reduces albuminuria by inhibition of PARP-induced podocyte apoptosis. METHODS: DM was induced in 4-week-old spontaneously hypertensive rats by streptozotocin. Mouse and human podocyte cell lines were cultured in normal or high-glucose conditions, with or without tempol and/or a PARP-1 inhibitor, PJ34. RESULTS: In diabetic rats, tempol treatment did not affect plasma glucose levels or systolic blood pressure. Albuminuria was higher in diabetic rats, and it was reduced by tempol. DM leads to an elevation of glomerular apoptotic cells and to podocyte loss; both were prevented by tempol treatment. DM increases the expression of poly(ADP-ribose)-modified proteins in isolated glomeruli, and it was reduced by tempol. In vitro, high glucose increased caspase-3 activity and led to a higher number of apoptotic cells that were prevented by tempol and the PARP-1 inhibitor. CONCLUSION: In DM, tempol reduces albuminuria associated with reduction of podocyte apoptosis and decreasing oxidative stress via PARP signaling.
Asunto(s)
Apoptosis/efectos de los fármacos , Óxidos N-Cíclicos/farmacología , Diabetes Mellitus Experimental/prevención & control , Podocitos/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transducción de Señal/efectos de los fármacos , Albuminuria/metabolismo , Albuminuria/prevención & control , Albuminuria/orina , Animales , Antioxidantes/farmacología , Western Blotting , Caspasa 3/metabolismo , Línea Celular Transformada , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Masculino , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/orina , Ratones , Estrés Oxidativo/efectos de los fármacos , Fenantrenos/farmacología , Podocitos/metabolismo , Podocitos/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Ratas , Ratas Endogámicas SHR , Marcadores de Spin , EstreptozocinaRESUMEN
In patients with diabetes, it has been suggested that physical exercise may reduce albuminuria and the progression of renal disease. However, the molecular mechanism by which physical exercise protects the kidney in diabetes remains poorly understood. The aim of the present study was to determine the contribution of muscle irisin secretion induced by aerobic physical exercise with the subsequent activation of AMPK for kidney protection under diabetic conditions. Aerobic physical exercise in rats protected the kidney in streptozotocin-induced diabetes. It reduced albuminuria, glomerular hypertrophy, and glomerular expression of collagen IV and fibronectin, as well as markers of kidney inflammation, when compared to sedentary diabetic rats. These effects were associated with elevation in muscle FNDC5/irisin and activity of AMPK in the diabetic kidney. However, the beneficial effects of exercise were lost when the diabetic rats were treated with CycloRGDyK, that in the bone it has been described as an irisin receptor blocker. In cultured human tubular (HK-2) cells, treatment with recombinant irisin counteracted the effect of high glucose in a dose-dependent manner. Irisin, per se, also activated AMPK in HK-2 cells. It is concluded that in diabetes, the renal protective effect of exercise may be mediated by the irisin/AMPK pathway.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Diabetes Mellitus Experimental , Nefropatías Diabéticas , Ejercicio Físico , Fibronectinas , Proteínas Quinasas Activadas por AMP/metabolismo , Albuminuria , Animales , Nefropatías Diabéticas/terapia , Fibronectinas/metabolismo , Humanos , Riñón/metabolismo , Condicionamiento Físico Animal , RatasRESUMEN
Green tea (GT), through its antioxidant properties, may be useful to treat or prevent human diseases. Because several lines of evidence suggest that oxidative stress contributes to the pathogenesis of diabetic nephropathy, we tested the hypothesis that GT prevents diabetes and hypertension-related renal oxidative stress, attenuating renal injury. Spontaneously hypertensive rats (SHR) with streptozotocin-induced diabetes and nondiabetic SHR were treated daily with tap water or freshly prepared GT (13.3 g/L). After 12 wk, the systolic blood pressure did not differ between treated and untreated nondiabetic or diabetic rats. However, body weight was less (P < 0.05) and glycemia was greater in diabetic SHR rats than in nondiabetic rats. Renal oxidative stress variables such as 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nitrotyrosine expression, NADPH oxidase-dependent superoxide generation, and the expression of renal cortex Nox4 were greater (P < 0.05) in diabetic rats that received water (DW) than in nondiabetic rats that received water (CW). The 8-OHdG and NADPH oxidase-dependent superoxide generation were significantly less in rats treated with GT. Nitrotyrosine and Nox4 expression were significantly less in diabetic rats that received GT (DGT) than in DW. Likewise, the indices of renal injury, albuminuria, and renal expression of collagen IV were significantly greater in DW than in CW. These differences were significantly less in DGT than in DW. GT reestablished the redox state and reduced the indicators of nephropathy without altering glycemia and blood pressure levels in diabetic SHR. These findings suggest that the consumption of GT may ameliorate nephropathy in diabetic hypertensive patients.
Asunto(s)
Camellia sinensis/química , Diabetes Mellitus Experimental/complicaciones , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Enfermedades Renales/prevención & control , NADPH Oxidasas/metabolismo , Extractos Vegetales/farmacología , Animales , Biomarcadores , Regulación hacia Abajo , Enfermedades Renales/complicaciones , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Estrés Oxidativo , Extractos Vegetales/química , Ratas , Ratas Endogámicas SHR , Superóxidos/metabolismoRESUMEN
AIMS: The presence of hypertension increases renal oxidative stress by increasing NADPH oxidase-dependent superoxide production and by decreasing antioxidant defense in the early stage of experimental diabetes mellitus (DM). In the present study, we investigated whether the administration of an antioxidant mimetic of the superoxide dismutase (SOD) (tempol) corrects the oxidative imbalance and oxidative stress-induced renal injury in the presence of DM and hypertension. METHODS: DM was induced in spontaneously hypertensive rats (SHR) by streptozotocin at 4 weeks of age. The diabetic rats either did or did not receive tempol for 20 days. Oxidative-stress parameters and indices of renal injury were evaluated. RESULTS: Tempol reestablished the imbalance in redox status induced by DM. It elevated the expression of renal antioxidant extracellular SOD, p < 0.0001; decreased (p = 0.049) the production of renal NADPH-dependent superoxide production, and diminished (p = 0.016) a marker of oxidative stress-induced DNA damage, 8-hydroxy-2'-deoxyguanosine. Reduction of oxidative stress markers was associated with reduction in renal damage parameters associated with DN. DM-induced albuminuria and elevation in renal expression of collagen IV were reduced to the level observed in control rats. CONCLUSION: We conclude that an imbalance in renal redox status is associated with markers of renal injury in the early stage of DM and hypertension. Antioxidant treatment reestablished the redox status and prevented oxidative stress-induced renal damage.
Asunto(s)
Antioxidantes/farmacología , Óxidos N-Cíclicos/farmacología , Nefropatías Diabéticas/tratamiento farmacológico , Hipertensión Renal/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Colágeno Tipo IV/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Progresión de la Enfermedad , Hipertensión Renal/metabolismo , Hipertensión Renal/patología , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Masculino , Imitación Molecular , NADPH Oxidasas/metabolismo , Ratas , Ratas Endogámicas SHR , Marcadores de Spin , Superóxido Dismutasa/metabolismoRESUMEN
Purpose: Outer blood retinal barrier breakdown is a neglected feature of diabetic retinopathy (DR). We demonstrated that the agonism of the δ opioid receptor (DOR) by epicatechin preserves the tight junction proteins in ARPE-19 cells under diabetic conditions. Presently, we aimed to evaluate the possible role of the DOR on the maintenance of tight junction of RPE layer and on the early markers of experimental DR. Methods: DR markers and external retinal tight junction proteins were evaluated in CL57B diabetic mice submitted to intravitreous injection of short hairpin RNA (shRNA)-DOR (108 transducing units [TU]/mL) treated or not with DOR agonist (0.05 g/animal/d of epicatechin in drinking water) for 16 weeks. The presence of DOR in human retina from postmortem eyes from diabetic and nondiabetic donors were also performed. Results: DOR is present in RPE layer and in neuro retina. The treatment with DOR agonist prevented the upregulation of the early markers of retinopathy (glial fibrillary acidic protein, VEGF) and the downregulation of pigment epithelium-derived factor, occludin, claudin-1, and zonula occludens-1 tight junction expressions. The silencing of DOR in retina of diabetic mice partially abolished the protective effects of epicatechin. In human retina specimens, DOR is present throughout the retina, similarly in nondiabetic and diabetic donors. Conclusions: This set of experiments strongly indicates that the DOR agonism preserves RPE tight junctions and reduces the early markers of retinopathy in model of diabetes. These novel findings designate DOR as a potential therapeutic tool to treat DR with preservation of the RPE tight junction proteins.
Asunto(s)
Catequina/farmacología , Diabetes Mellitus Experimental/prevención & control , Retinopatía Diabética/prevención & control , Receptores Opioides delta/agonistas , Epitelio Pigmentado de la Retina/metabolismo , Uniones Estrechas/metabolismo , Anciano , Animales , Glucemia/metabolismo , Western Blotting , Claudina-1/metabolismo , Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Impedancia Eléctrica , Proteínas del Ojo/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Factores de Crecimiento Nervioso/metabolismo , Ocludina/metabolismo , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Opioides delta/metabolismo , Serpinas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
BACKGROUND/AIMS: Previous partial hepatectomy (HPTX) can attenuate glycerol-induced acute kidney injury (Gly-AKI). The aim of this study was to explore the pathophysiological mechanisms and the role of hepatocyte growth factor (HGF) in kidney protection. METHODS: Rats were subjected to HPTX 24 h before glycerol administration. Renal function, acute tubular necrosis, apoptosis, leukocyte infiltration, and the expression of HGF, c-met, monocyte chemoattractant protein-1, interleukin-1beta, and heme oxygenase-1 were evaluated 24 h after glycerol injection. The regenerative response was analyzed from 6 to 72 h after glycerol injection (BrdU incorporation). In a separate series of experiments, Gly-AKI+HPTX rats were treated with anti-HGF antibody. RESULTS: Gly-AKI+HPTX rats showed an increased expression of renal HGF and c-met as well as an improved creatinine clearance and reduced acute tubular necrosis and apoptosis, cytokine expression, and leukocyte infiltration. The regenerative response was less intense 24 and 72 h after glycerol administration in this group. The anti-HGF treatment disclosed an important role of HGF in the reduction of tubular injury, particularly apoptosis. Overexpression of heme oxygenase-1 was observed in Gly-AKI+HPTX rats, but was not associated with HPTX-induced renal protection. CONCLUSION: We conclude that Gly-AKI+HPTX rats have a reduced susceptibility to renal injury instead of an increased regenerative response and that endogenous HGF overexpression is responsible for suppression of tubular apoptosis.
Asunto(s)
Glicerol/toxicidad , Hepatectomía , Factor de Crecimiento de Hepatocito/fisiología , Necrosis Tubular Aguda/prevención & control , Proteínas Proto-Oncogénicas c-met/fisiología , Animales , Apoptosis/efectos de los fármacos , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/genética , Creatinina/sangre , Regulación de la Expresión Génica , Hemo Oxigenasa (Desciclizante)/biosíntesis , Hemo Oxigenasa (Desciclizante)/genética , Factor de Crecimiento de Hepatocito/biosíntesis , Factor de Crecimiento de Hepatocito/genética , Interleucina-1/biosíntesis , Interleucina-1/genética , Riñón/metabolismo , Riñón/fisiopatología , Necrosis Tubular Aguda/inducido químicamente , Necrosis Tubular Aguda/metabolismo , Necrosis Tubular Aguda/patología , Necrosis Tubular Aguda/cirugía , Macrófagos/patología , Proteínas Proto-Oncogénicas c-met/biosíntesis , Proteínas Proto-Oncogénicas c-met/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Regeneración , Linfocitos T/patologíaRESUMEN
The aim of this study was to evaluate the effect of prevention of hypertension on glomerular hypertrophy, renal cell replication and accumulation of glomerular fibronectin in a model of genetic hypertension and experimental diabetes. Four-week-old streptozotocin induced spontaneously hypertensive rats (SHR) were randomized for no treatment, or for treatment with captopril, losartan or triple therapy (hydrochlorothiazide, reserpine and hydralazine) for 20 days. Increase in systolic blood pressure was equally prevented by captopril (118+/-15 mmHg), losartan (111+/-9) and triple therapy (112+/-14, p<0.0001). Glomerular size was higher (p<0.005) in diabetic SHR (27,300+/-2130 microm(2)) compared with non-diabetic SHR (23,800+/-307). The antihypertensive therapy with captopril (23,900+/-175), losartan (23,800+/-120), and triple therapy (23,400+/-210) prevented the glomerular enlargement in diabetic SHR. Glomerular expression of fibronectin was increased in diabetic SHR (7.61+/-1.22 densitometric unit) as compared to the controls (2.27+/-2.15, p<0.0001), and was decreased (p<0.0001 vs diabetic SHR) with captopril (2.49+/-1.42), losartan (1.57+/-1.1) and triple therapy (2.04+/-1.42). The number of replicating glomerular cell significantly decreased in diabetic SHR and it was restored by all three antihypertensive regimes. The glomerular expression of p27(Kip1) was increased in diabetic SHR but it was not modified by antihypertensive treatment. Strict blood pressure control, in diabetic SHR independently of the class of antihypertensive agent, restores glomerular hypertrophy and renal cellular replication, and prevents the increment in glomerular fibronectin.
Asunto(s)
Presión Sanguínea/fisiología , Diabetes Mellitus Experimental/fisiopatología , Hipertensión/fisiopatología , Glomérulos Renales/patología , Animales , Antihipertensivos/uso terapéutico , Presión Sanguínea/efectos de los fármacos , Captopril/uso terapéutico , Diabetes Mellitus Experimental/tratamiento farmacológico , Hipertensión/patología , Inmunohistoquímica , Glomérulos Renales/efectos de los fármacos , Ratas , Ratas Endogámicas SHRRESUMEN
PURPOSE: Müller cells (MCs) are a major source of VEGF in diabetic retinopathy (DR). Vascular endothelial growth factor is the main therapeutic target for treating DR. This study aimed to investigate whether autophagy is involved in MC response under high glucose (HG). METHODS: Rat retinal Müller cells (rMCs) were exposed to normal or high glucose in and out of presence of pharmacologic inhibitors and activators and small interfering RNA (siRNA) for p62/SQTSM1 for 24 hours. RESULTS: High glucose induces increase of early and late autophagic markers, accumulation of p62/SQTSM1 and endoplasmic reticulum (ER) stress response associated with apoptosis augmentation (P < 0.01). The inhibition of autophagy in HG leads to higher rMC apoptotic rate (P < 0.001). By silencing the p62/SQTSM1, ER stress is ameliorated (p<0.0001), preventing apoptosis. Retinal MCs in HG treated with rapamycin (mTOR inhibitor) show autophagy machinery activation and reestablishment of cargo degradation, protecting cells from apoptosis (P < 0.0001). Rapamycin improves lysosomal proteolytic activity by improving cathepsin L activity restoring autophagic cargo degradation, and preventing increased VEGF release (P < 0.0001). In experimental model of diabetes, Beclin-1 and p62/SQTSM-1 were found to be marked increased in retinas from diabetic Wystar Kyoto rats compared with control group (P < 0.003) with reduction of cathepsin L activity. CONCLUSIONS: High glucose upregulates autophagy but accumulates p62/SQTSM1 cargo due to lysosomal dysfunction, leading to massive VEGF release and cell death of rMCs. Lysosomal impairment and autophagic dysfunction are early events present in the pathogenesis of diabetic retinopathy (DR). This might be valuable for developing a novel therapeutic strategy to treat DR.
Asunto(s)
Autofagia/fisiología , Diabetes Mellitus Experimental , Retinopatía Diabética/patología , Regulación de la Expresión Génica , ARN Interferente Pequeño/genética , Retina/metabolismo , Proteína Sequestosoma-1/genética , Animales , Apoptosis , Autofagia/efectos de los fármacos , Western Blotting , Células Cultivadas , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Células Ependimogliales/metabolismo , Células Ependimogliales/ultraestructura , Glucosa/farmacología , Microscopía Electrónica de Transmisión , Estrés Oxidativo , ARN Interferente Pequeño/metabolismo , Ratas , Retina/patología , Proteína Sequestosoma-1/biosíntesis , Edulcorantes/farmacologíaRESUMEN
Prior research has shown that in experimental diabetes mellitus, green tea reduces albuminuria by decreasing podocyte apoptosis through activation of the WNT pathway. We investigated the effect of green tea polyphenols (GTP) on residual albuminuria of diabetic subjects with nephropathy. We conducted a randomised, double-blind study in 42 diabetic subjects with a urinary albumin-creatinine ratio (UACR) >30 mg/g, despite administration of the maximum recommended dose of renin-angiotensin (RAS) inhibition. Patients were randomly assigned to two equal groups to receive either GTP (containing 800 mg of epigallocatechin gallate, 17 with type 2 diabetes and 4 with type 1 diabetes) or placebo (21 with type 2 diabetes) for 12 weeks. Treatment with GTP reduced UACR by 41%, while the placebo group saw a 2% increase in UACR (p = 0.019). Podocyte apoptosis (p = 0.001) and in vitro albumin permeability (p < 0.001) were higher in immortalized human podocytes exposed to plasma from diabetic subjects compared to podocytes treated with plasma from normal individuals. In conclusion, GTP administration reduces albuminuria in diabetic patients receiving the maximum recommended dose of RAS. Reduction in podocyte apoptosis by activation of the WNT pathway may have contributed to this effect.
Asunto(s)
Albuminuria/tratamiento farmacológico , Catequina/análogos & derivados , Nefropatías Diabéticas/tratamiento farmacológico , Pisum sativum/química , Polifenoles/administración & dosificación , Anciano , Albuminuria/metabolismo , Albuminuria/patología , Apoptosis/efectos de los fármacos , Catequina/administración & dosificación , Catequina/química , Células Cultivadas , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Podocitos/metabolismo , Podocitos/patología , Polifenoles/químicaRESUMEN
Bone marrow-derived cells were demonstrated to improve organ function, but the lack of cell retention within injured organs suggests that the protective effects are due to factors released by the cells. Herein, we tested cell therapy using early outgrowth cells (EOCs) or their conditioned media (CM) to protect the retina of diabetic animal models (type 1 and type 2) and assessed the mechanisms by in vitro study. Control and diabetic (db/db) mice (8 weeks of age) were randomized to receive a unique intravenous injection of 5×105EOCs or 0.25 ml thrice weekly tail-vein injections of 10x concentrated CM and Wystar Kyoto rats rendered diabetic were randomized to receive 0.50 ml thrice weekly tail-vein injections of 10x concentrated CM. Four weeks later, the animals were euthanized and the eyes were enucleated. Rat retinal Müller cells (rMCs) were exposed for 24 h to high glucose (HG), combined or not with EOC-conditioned medium (EOC-CM) from db/m EOC cultures. Diabetic animals showed increase in diabetic retinopathy (DR) and oxidative damage markers; the treatment with EOCs or CM infusions significantly reduced this damage and re-established the retinal function. In rMCs exposed to diabetic milieu conditions (HG), the presence of EOC-CM reduced reactive oxygen species production by modulating the NADPH-oxidase 4 system, thus upregulating SIRT1 activity and deacetylating Lys-310-p65-NFκB, decreasing GFAP and VEGF expressions. The antioxidant capacity of EOC-CM led to the prevention of carbonylation and nitrosylation posttranslational modifications on the SIRT1 molecule, preserving its activity. The pivotal role of SIRT1 on the mode of action of EOCs or their CM was also demonstrated on diabetic retina. These findings suggest that EOCs are effective as a form of systemic delivery for preventing the early molecular markers of DR and its conditioned medium is equally protective revealing a novel possibility for cell-free therapy for the treatment of DR.
Asunto(s)
Células de la Médula Ósea/metabolismo , Medios de Cultivo Condicionados/farmacología , Sustancias Protectoras/farmacología , Retina/efectos de los fármacos , Retina/metabolismo , Animales , Biomarcadores , Glucemia , Línea Celular Transformada , Retinopatía Diabética/diagnóstico , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/genética , Retinopatía Diabética/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía , Células Ependimogliales/metabolismo , Masculino , Ratones , Estrés Oxidativo , Sustancias Protectoras/administración & dosificación , Ratas , Especies Reactivas de Oxígeno , Retina/patología , Transducción de Señal , Sirtuina 1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
PURPOSE: Retinal pigment epithelium cells, along with tight junction (TJ) proteins, constitute the outer blood retinal barrier (BRB). Contradictory findings suggest a role for the outer BRB in the pathogenesis of diabetic retinopathy (DR). The aim of this study was to investigate whether the mechanisms involved in these alterations are sensitive to nitrosative stress, and if cocoa or epicatechin (EC) protects from this damage under diabetic (DM) milieu conditions. METHODS: Cells of a human RPE line (ARPE-19) were exposed to high-glucose (HG) conditions for 24 hours in the presence or absence of cocoa powder containing 0.5% or 60.5% polyphenol (low-polyphenol cocoa [LPC] and high-polyphenol cocoa [HPC], respectively). RESULTS: Exposure to HG decreased claudin-1 and occludin TJ expressions and increased extracellular matrix accumulation (ECM), whereas levels of TNF-α and inducible nitric oxide synthase (iNOS) were upregulated, accompanied by increased nitric oxide levels. This nitrosative stress resulted in S-nitrosylation of caveolin-1 (CAV-1), which in turn increased CAV-1 traffic and its interactions with claudin-1 and occludin. This cascade was inhibited by treatment with HPC or EC through δ-opioid receptor (DOR) binding and stimulation, thereby decreasing TNF-α-induced iNOS upregulation and CAV-1 endocytosis. The TJ functions were restored, leading to prevention of paracellular permeability, restoration of resistance of the ARPE-19 monolayer, and decreased ECM accumulation. CONCLUSIONS: The detrimental effects on TJs in ARPE-19 cells exposed to DM milieu occur through a CAV-1 S-nitrosylation-dependent endocytosis mechanism. High-polyphenol cocoa or EC exerts protective effects through DOR stimulation.
Asunto(s)
Cacao/química , Caveolina 1/metabolismo , Endocitosis/fisiología , Polifenoles/farmacología , Receptores Opioides/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Uniones Estrechas/metabolismo , Animales , Barrera Hematorretinal , Western Blotting , Línea Celular , Claudina-1/metabolismo , Dextranos/metabolismo , Impedancia Eléctrica , Técnica del Anticuerpo Fluorescente Indirecta , Glucosa/farmacología , Humanos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitrosación , Ocludina/metabolismo , Permeabilidad , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Porcinos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The aims of the present study were to investigate, in diabetes mellitus (DM), the mechanism of NOX4 up-regulation, its link with 5' adenosine monophosphate-activated protein kinase (AMPK) inactivation and transforming growth factor (TGF) ß-1 signaling in determining the accumulation of kidney extracellular matrix (ECM), and the possible action of cocoa enriched with polyphenols (CH) in these events. After 16 weeks of DM, spontaneously hypertensive rats showed increased kidney TGFß-1 levels and expression of phosphorylated smad2, collagen IV and fibronectin in parallel with elevated NOX4 expression and reduced phosphorylated AMPK. CH treatment in diabetic rats prevented all of these abnormalities. In immortalized human mesangial cells exposed to high glucose (HG), or TGFß-1, CH, nicotinamide adenine dinucleotide phosphate blocker, or silencing NOX4 ameliorated enhanced phosphorylated smad2 and collagen IV. Reduction in phosphorylated AMPK induced by HG or TGFß-1 was ameliorated by CH or activation of AMPK, which reduced phosphorylation of smad2 and collagen IV via reduction in NOX4 expression. The effects of CH were abolished by AMPK blockade. These results suggest that inactivation in AMPK leads to NOX4 up-regulation, activation of TGFß-1 signaling and increased ECM accumulation. Additionally, increased TGF-ß1 per se leads to the amplification of ECM production by reducing AMPK and promoting the activation of NOX4. It is suggested that the activation of AMPK by CH followed by reduction in NOX4/TGFß-1 signaling may have a therapeutic potential in diabetic nephropathy.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Nefropatías Diabéticas/prevención & control , NADPH Oxidasas/efectos de los fármacos , Polifenoles/farmacología , Animales , Cacao , NADPH Oxidasa 4 , Ratas Endogámicas SHR , Factor de Crecimiento Transformador beta1/biosíntesis , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The ability of nano-assisted laser desorption-ionization mass spectrometry imaging (NALDI-IMS) to provide selective chemical monitoring with appropriate spatial distribution of a low molecular drug in a biological tissue was investigated. NALDI-IMS is a matrix-free laser desorption ionization (LDI) protocol based on imprinting of tissue constituents on a nanostructured surface. Using the accumulation of theobromine in rat kidney as a model, NALDI-IMS was found to provide well-resolved images of the special distribution of this low molecular weight (MW) drug in tissue.
Asunto(s)
Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Teobromina/análisis , Teobromina/química , Animales , Riñón/química , Riñón/metabolismo , Masculino , Peso Molecular , RatasRESUMEN
PURPOSE: Diabetic retinopathy (DR) is associated with nitrosative stress. The purpose of this study was to evaluate the beneficial effects of S-nitrosoglutathione (GSNO) eye drop treatment on an experimental model of DR. METHODS: Diabetes (DM) was induced in spontaneously hypertensive rats (SHR). Treated animals received GSNO eye drop (900 nM or 10 µM) twice daily in both eyes for 20 days. The mechanisms of GSNO effects were evaluated in human RPE cell line (ARPE-19). RESULTS: In animals with DM, GSNO decreased inducible nitric oxide synthase (iNOS) expression and prevented tyrosine nitration formation, ameliorating glial dysfunction measured with glial fibrillary acidic protein, resulting in improved retinal function. In contrast, in nondiabetic animals, GSNO induced oxidative/nitrosative stress in tissue resulting in impaired retinal function. Nitrosative stress was present markedly in the RPE layer accompanied by c-wave dysfunction. In vitro study showed that treatment with GSNO under high glucose condition counteracted nitrosative stress due to iNOS downregulation by S-glutathionylation, and not by prevention of decreased GSNO and reduced glutathione levels. This posttranslational modification probably was promoted by the release of oxidized glutathione through GSNO denitrosylation via GSNO-R. In contrast, in the normal glucose condition, GSNO treatment promoted nitrosative stress by NO formation. CONCLUSIONS: In this study, a new therapeutic modality (GSNO eye drop) targeting nitrosative stress by redox posttranslational modification of iNOS was efficient against early damage in the retina due to experimental DR. The present work showed the potential clinical implications of balancing the S-nitrosoglutathione/glutathione system in treating DR.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo II/metabolismo , S-Nitrosoglutatión/farmacología , Análisis de Varianza , Animales , Biomarcadores/metabolismo , Línea Celular , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Glutatión/metabolismo , Humanos , Donantes de Óxido Nítrico/uso terapéutico , Soluciones Oftálmicas/farmacología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , S-Nitrosoglutatión/uso terapéutico , Tirosina/análogos & derivados , Tirosina/metabolismo , Regulación hacia ArribaRESUMEN
Diabetes and hypertension frequently coexist and constitute the most notorious combination for the pathogenesis of DR. Large clinical trials have clearly demonstrated that tight control of glycaemia and/or blood pressure significantly reduces the incidence and progression of DR. The mechanism by which hypertension interacts with diabetes to exacerbate the retinal disease is not completely understood. From experimental studies, increasing evidence demonstrates that chronic inflammation and oxidative stress are involved. In the present review, we summarize data obtained from our research along with those from other groups to better understand the role of hypertension in the pathogenesis of DR. It is suggested that oxidative stress and inflammation may be common denominators of retinal damage in the presence of hypertension in diabetic patients.
Asunto(s)
Diabetes Mellitus/fisiopatología , Retinopatía Diabética/etiología , Hipertensión/complicaciones , Animales , Glucemia , Presión Sanguínea , Retinopatía Diabética/patología , Progresión de la Enfermedad , Humanos , Hipertensión/fisiopatología , Inflamación/patología , Estrés OxidativoRESUMEN
The current study investigated the potential of green tea (GT) to improve uncoupling of endothelial nitric oxide synthase (eNOS) in diabetic conditions. In rats with streptozotocin-induced diabetes, nitric oxide (NO) bioavailability was reduced by uncoupling eNOS, characterized by a reduction in tetrahydrobiopterin (BH(4)) levels and a decrease in the eNOS dimer-to-monomer ratio. GT treatment ameliorated these abnormalities. Moreover, immortalized human mesangial cells (ihMCs) exposed to high glucose (HG) levels exhibited a rise in reactive oxygen species (ROS) and a decline in NO levels, which were reversed with GT. BH(4) and the activity of guanosine triphosphate cyclohydrolase I decreased in ihMCs exposed to HG and was normalized by GT. Exogenous administration of BH(4) in ihMCs reversed the HG-induced rise in ROS and the decline in NO production. However, coadministration of GT with BH(4) did not result in a further reduction in ROS production, suggesting that reduced ROS with GT was indeed secondary to uncoupled eNOS. In summary, GT reversed the diabetes-induced reduction of BH(4) levels, ameliorating uncoupling eNOS, and thus increasing NO bioavailability and reducing oxidative stress, two abnormalities that are involved in the pathogenesis of diabetic nephropathy.
Asunto(s)
Biopterinas/análogos & derivados , Camellia sinensis , Diabetes Mellitus Experimental/enzimología , Células Mesangiales/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Animales , Antioxidantes/farmacología , Biopterinas/análisis , Biopterinas/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/tratamiento farmacológico , GTP Ciclohidrolasa/metabolismo , Humanos , Células Mesangiales/enzimología , Óxido Nítrico/análisis , Óxido Nítrico/biosíntesis , Estrés Oxidativo/efectos de los fármacos , Ratas , Ratas Endogámicas SHR , Especies Reactivas de Oxígeno/análisis , TéRESUMEN
Spironolactone (SPR), a mineralocorticoid receptor blocker, diminishes hyperglycemia-induced reduction in glucose-6-phosphate dehydrogenase (G6PD) activity, improving oxidative stress damage. This study investigated whether SPR ameliorates nephropathy by increasing G6PD activity and reducing oxidative stress in spontaneously hypertensive diabetic rats (SHRs). The streptozotocin-induced diabetic rats received or not SPR 50 mg/kg per day, for eight weeks. A human mesangial cell line was cultured in normal or high glucose conditions, with or without SPR, for 24 h. Plasma glucose levels and systolic blood pressure were unaltered by diabetes or by SPR treatment. Albuminuria, fibronectin expression, 8-OHdG urinary levels, lipid peroxidation and p47phox expression were higher in the diabetic rats compared with the control and were reduced by SPR. The antioxidant GSH/GSSG ratio was reduced in the diabetic rats and the treatment reestablished it. Diabetes-induced SGK1 up-regulation was inhibited by SPR. Reactive oxygen species (ROS) and superoxide production induced by NADPH oxidase were increased by hyperglycemia and high glucose, in vivo and in vitro, respectively, and were reduced with SPR. Hyperglycemia and high glucose decreased G6PD activity, which was restored with SPR. These results suggest that SPR ameliorates nephropathy in diabetic SHRs by restoring G6PD activity and diminishes oxidative stress without affecting glycaemia and blood pressure.