RESUMEN
Upper urinary tract obstruction results in tubulointerstitial fibrosis and a progressive decline in renal function. Although several inflammatory mediators have been implicated in the pathophysiology of renal obstruction, the contribution of TNF-alpha to obstruction-induced fibrosis and renal dysfunction has not been thoroughly evaluated. To study this, male Sprague-Dawley rats were subjected to left unilateral ureteral obstruction vs. sham operation. Rats received either vehicle or a pegylated form of soluble TNF receptor type 1 (PEG-sTNFR1) every 84 h. The kidneys were harvested 1, 3, or 7 days postoperatively, and tissue samples were analyzed for TNF-alpha expression (ELISA), macrophage infiltration (ED-1 staining), transforming growth factor-beta(1) expression (ELISA, RT-PCR), collagen I and IV activity (Western Blot, immunohistochemistry), alpha-smooth muscle actin accumulation (immunohistochemistry, Western blot analysis), and angiotensinogen expression (Western blot). In a separate arm, the glomerular filtration rate (inulin clearance) of rats subjected to unilateral ureteral obstruction in the presence of either vehicle or PEG-sTNFR1 was determined. Renal obstruction induced increased tissue TNF-alpha and transforming growth factor-beta(1) levels, collagen I and IV activity, interstitial volume, alpha-smooth muscle actin accumulation, angiotensinogen expression, and renal dysfunction, whereas treatment with PEG-sTNFR1 significantly reduced each of these markers of renal fibrosis. These results demonstrate that TNF-alpha mediates obstruction-induced renal fibrosis and identify TNF-alpha neutralization as a potential therapeutic option for the amelioration of obstruction-induced renal injury.
Asunto(s)
Enfermedades Renales/patología , Receptores Tipo I de Factores de Necrosis Tumoral/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/patología , Actinas/biosíntesis , Angiotensinógeno/metabolismo , Animales , Biomarcadores/metabolismo , Western Blotting , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Fibrosis/etiología , Fibrosis/patología , Semivida , Inmunohistoquímica , Corteza Renal/citología , Corteza Renal/inmunología , Corteza Renal/metabolismo , Enfermedades Renales/etiología , Cinética , Macrófagos/metabolismo , Masculino , Peso Molecular , Polietilenglicoles/química , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Receptores Tipo I de Factores de Necrosis Tumoral/química , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/uso terapéutico , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Solubilidad , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Uréter/cirugía , Obstrucción Ureteral/etiologíaRESUMEN
Obstruction of the upper urinary tract induces a progressive loss in renal mass through apoptotic renal cell death. Although TNF-alpha has been implicated in ischemia-reperfusion-induced apoptotic renal cell death, its role in obstructive renal cell apoptosis remains unknown. To study this, male Sprague-Dawley rats were subjected to left unilateral ureteral obstruction vs. sham operation. Twenty-four hours before surgery and every 84 h thereafter, rats received either vehicle or a pegylated form of soluble TNF receptor type 1 (PEG-sTNFR1). The kidneys were harvested 1, 3, or 7 days postoperatively, and tissue samples were subsequently analyzed for TNF-alpha (ELISA, RT-PCR), Fas ligand (RT-PCR), apoptosis (TUNEL, ELISA), and caspase 8 and 3 activity (Western blot). Renal obstruction induced increased tissue TNF-alpha and Fas ligand mRNA levels, TNF-alpha protein production, apoptotic renal tubular cell death, and elevated caspase 8 and 3 activity, whereas treatment with PEG-sTNFR1 significantly reduced obstruction-induced TNF-alpha production, renal tubular cell apoptosis, and caspase activity. PEG-sTNFR1 did not significantly alter Fas ligand expression. These results demonstrate that TNF-alpha mediates obstruction-induced renal tubular cell apoptosis and proapoptotic signaling and identify TNF-alpha neutralization as a potential therapeutic option for the amelioration of obstruction-induced renal injury.
Asunto(s)
Apoptosis/genética , Apoptosis/fisiología , Túbulos Renales/patología , Factor de Necrosis Tumoral alfa/farmacología , Obstrucción Ureteral/complicaciones , Animales , Proteína Ligando Fas , Humanos , Inflamación , Masculino , Glicoproteínas de Membrana/biosíntesis , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Obstrucción Ureteral/patología , Obstrucción Ureteral/veterinariaRESUMEN
Ischemia causes renal tubular cell loss through apoptosis; however, the mechanisms of this process remain unclear. Using the renal tubular epithelial cell line LLC-PK(1), we developed a model of simulated ischemia (SI) to investigate the role of p38 MAPK (mitogen-activated protein kinase) in renal cell tumor necrosis factor-alpha (TNF-alpha) mRNA production, protein bioactivity, and apoptosis. Results demonstrate that 60 min of SI induced maximal TNF-alpha mRNA production and bioactivity. Furthermore, 60 min of ischemia induced renal tubular cell apoptosis at all substrate replacement time points examined, with peak apoptotic cell death occurring after either 24 or 48 h. p38 MAPK inhibition abolished TNF-alpha mRNA production and TNF-alpha bioactivity, and both p38 MAPK inhibition and TNF-alpha neutralization (anti-porcine TNF-alpha antibody) prevented apoptosis after 60 min of SI. These results constitute the initial demonstration that 1) renal tubular cells produce TNF-alpha mRNA and biologically active TNF-alpha and undergo apoptosis in response to SI, and 2) p38 MAPK mediates renal tubular cell TNF-alpha production and TNF-alpha-dependent apoptosis after SI.
Asunto(s)
Apoptosis/fisiología , Isquemia/fisiopatología , Túbulos Renales/irrigación sanguínea , Túbulos Renales/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Activación Enzimática , Túbulos Renales/patología , Células LLC-PK1 , ARN Mensajero/metabolismo , Porcinos , Factores de Tiempo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
PURPOSE: Renal ischemia-reperfusion (IR) injury is a devastating clinical problem. While effective animal models have been developed to investigate this condition, they are limited by differential renal cell inflammatory mediator production and heterogeneous cell sensitivity to ischemia. We therefore developed an in vitro model of renal tubular cell ischemia that simulates the cellular injury observed in animal models of renal IR injury. MATERIALS AND METHODS: Using the established renal tubular cell line, LLC-PK1, simulated ischemia was induced by immersing the cellular monolayer in mineral oil. The effect of simulated ischemia on renal tubular cells was then determined by measuring the time course of TNF-alpha protein expression (ELISA), TNF-alpha mRNA induction (RT-PCR), and renal tubular cell apoptosis (TUNEL). RESULTS: Maximal TNF-alpha protein expression occurs following 60 min of simulated ischemia and 2 h of substrate replacement (reimmersion in media), and maximal TNF-alpha mRNA induction occurs following 60 min of simulated ischemia. Cellular apoptosis peaks following 60 min of simulated ischemia and 24 h of reperfusion. CONCLUSION: The time course of TNF-alpha production and apoptosis induction in this model closely parallels the time course for these markers in vivo. This study constitutes the initial demonstration that an in vitro oil immersion model of ischemia simulates the cellular injury (TNF-alpha production and apoptosis) observed in animal models of renal ischemia-reperfusion. This model may be used to study cellular mechanisms of IR in the absence of the systemic confounding variables.