RESUMEN
For 40 years indirect measurements of the glomerular sieving coefficient of albumin yielded very low values. The first direct measurement by 2-photon microscopy by Russo et al (Kidney Int (2007) 71, 504-513) gives values 50-times higher. This demonstrated that relatively large quantities of albumin are normally filtered based on size selectivity alone. Most of this albumin is retrieved and returned to the blood supply. These new discoveries represent a paradigm shift in our understanding of albumin processing by the kidney. They also serve to explain several anomalous aspects of previous studies on glomerular filtration and mechanism of albuminuria and support the fact that glomerular charge selectivity is not a major factor controlling glomerular permselectivity.
Asunto(s)
Albúminas/metabolismo , Artefactos , Riñón/química , Riñón/fisiología , Albúminas/ultraestructura , Albuminuria/metabolismo , Albuminuria/fisiopatología , Animales , Sitios de Unión , Transporte Biológico , Filtración , Tasa de Filtración Glomerular , Riñón/fisiopatología , Glomérulos Renales/fisiología , Túbulos Renales/metabolismo , Túbulos Renales/patología , Cinética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Síndrome Nefrótico/metabolismo , Síndrome Nefrótico/patologíaRESUMEN
The introduction of two-photon microscopy, along with the development of new fluorescent probes and innovative computer software, has advanced the study of intracellular and intercellular processes in the tissues of living organisms. Researchers can now determine the distribution, behavior, and interactions of labeled chemical probes and proteins in live kidney tissue in real time without fixation artifacts. Chemical probes, such as fluorescently labeled dextrans, have extended our understanding of dynamic events with subcellular resolution. To accomplish expression of specific proteins in vivo, cDNAs of fluorescently labeled proteins have been cloned into adenovirus vectors and infused by micropuncture to induce proximal tubule cell infection and protein expression. The localization and intensity of the expressed fluorescent proteins can be observed repeatedly at different time points allowing for enhanced quantitative analysis while limiting animal use. Optical sections of images acquired with the two-photon microscope can be 3-D reconstructed and quantified with Metamorph, Voxx, and Amira software programs.
Asunto(s)
Isquemia/metabolismo , Riñón/metabolismo , Microscopía Confocal , Microscopía de Fluorescencia por Excitación Multifotónica , Microscopía por Video , Adenoviridae/genética , Animales , Agregación Eritrocitaria , Colorantes Fluorescentes/metabolismo , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Interpretación de Imagen Asistida por Computador , Imagenología Tridimensional , Isquemia/sangre , Isquemia/patología , Riñón/irrigación sanguínea , Riñón/patología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Óptica y Fotónica , Programas Informáticos , Factores de TiempoRESUMEN
Acute Kidney Injury (AKI) is a complex disorder for which currently there is no accepted definition. We describe an initiative to develop uniform standards for defining and classifying AKI and establish a forum for multidisciplinary interaction to improve care for patients with, or at risk for AKI. Members representing key societies in critical care and nephrology along with additional experts in adult and pediatric AKI participated in a 2-day conference in Amsterdam in September 2005 to draft consensus recommendations for diagnosing and staging AKI. This report describes the proposed diagnostic and staging criteria for AKI and the formation of a multidisciplinary collaborative network.
Asunto(s)
Lesión Renal Aguda/diagnóstico , Lesión Renal Aguda/clasificación , HumanosRESUMEN
Secretion of cationic drugs and endogenous metabolites is a major function of the kidney accomplished by tubular organic cation transport systems. A cationic styryl dye (ASP(+)) was developed as a fluorescent substrate for renal organic cation transporters. The dye was injected intravenously and continuously monitored in externalized rat kidneys by time-resolved two-photon laser scanning microscopy. To investigate changes in transport activity, cimetidine, a competitive inhibitor of organic cation transport was co-injected with ASP(+). Shortly after injection, fluorescence increased in peritubular capillaries. Simultaneously, fluorescence was transiently found at the basolateral membrane of the proximal and distal tubules at a higher intensity and shorter wavelength indicating membrane association of ASP(+). Subsequently, intracellular fluorescence increased steeply within 10 s. In the proximal tubules, intracellular fluorescence decreased by 50% within 5 min, while in the distal tubules the fluorescence decreased by only 5% within the same time frame. Intracellular uptake of ASP(+) into proximal tubules was significantly reduced by cimetidine. Our studies show that organic cation transport of the kidney can be visualized in vivo by two-photon laser scanning microscopy.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Riñón/metabolismo , Microscopía Confocal , Microscopía de Fluorescencia por Excitación Multifotónica , Microscopía por Video , Proteínas de Transporte de Catión Orgánico/metabolismo , Compuestos de Piridinio/metabolismo , Animales , Transporte Biológico , Cationes/metabolismo , Cimetidina/farmacología , Colorantes Fluorescentes/administración & dosificación , Inyecciones Intravenosas , Riñón/irrigación sanguínea , Riñón/efectos de los fármacos , Túbulos Renales Distales/metabolismo , Túbulos Renales Proximales/metabolismo , Masculino , Microcirculación/metabolismo , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Compuestos de Piridinio/administración & dosificación , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
The origin of albuminuria remains controversial owing to difficulties in quantifying the actual amount of albumin filtered by the kidney. Here we use fluorescently labeled albumin, together with the powerful technique of intravital 2-photon microscopy to show that renal albumin filtration in non-proteinuric rats is approximately 50 times greater than previously measured and is followed by rapid endocytosis into proximal tubule cells (PTCs). The endocytosed albumin appears to undergo transcytosis in large vesicles (500 nm in diameter), identified by immunogold staining of endogenous albumin by electron microscopy, to the basolateral membrane where the albumin is disgorged back to the peritubular blood supply. In nephrotic rats, the rate of uptake of albumin by the proximal tubule (PT) is decreased. This is consistent with reduced expression of clathrin, megalin, and vacuolar H(+)-ATPase A subunit, proteins that are critical components of the PT endocytotic machinery. These findings strongly support the paradigm-shifting concept that the glomerular filter normally leaks albumin at nephrotic levels. Albuminuria does not occur as this filtered albumin load is avidly bound and retrieved by PTCs. Dysfunction of this retrieval pathway leads to albuminuria. Thus, restoration of the defective endocytotic and processing function of PT epithelial cells might represent an effective strategy to limit urinary albumin loss, at least in some types of nephrotic syndrome.
Asunto(s)
Albúminas/metabolismo , Glomérulos Renales/metabolismo , Túbulos Renales Proximales/metabolismo , Síndrome Nefrótico/metabolismo , Albuminuria/metabolismo , Albuminuria/patología , Albuminuria/fisiopatología , Animales , Clatrina/genética , Clatrina/metabolismo , Endocitosis/fisiología , Regulación de la Expresión Génica/fisiología , Tasa de Filtración Glomerular/fisiología , Glomérulos Renales/patología , Glomérulos Renales/fisiopatología , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/fisiopatología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Microscopía Electrónica/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Síndrome Nefrótico/patología , Síndrome Nefrótico/fisiopatología , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Ratas , Ratas WistarRESUMEN
Disruption of the actin cytoskeleton in proximal tubule cells is a key pathophysiological factor in acute renal failure. To investigate dynamic alterations of the actin cytoskeleton in live proximal tubule cells, LLC-PK(10) cells were transfected with an enhanced yellow fluorescence protein (EYFP)-actin construct, and a clone with stable EYFP-actin expression was established. Confluent live cells were studied by confocal microscopy under physiological conditions or during ATP depletion of up to 60 min. Immunoblots of stable transfected LLC-PK(10) cells confirmed the presence of EYFP-actin, accounting for 5% of total actin. EYFP-actin predominantly incorporated in stress fibers, i.e., cortical and microvillar actin as shown by excellent colocalization with Texas red phalloidin. Homogeneous cytosolic distribution of EYFP-actin indicated colocalization with G-actin as well. Beyond previous findings, we observed differential subcellular disassembly of F-actin structures: stress fibers tagged with EYFP-actin underwent rapid and complete disruption, whereas cortical and microvillar actin disassembled at slower rates. In parallel, ATP depletion induced the formation of perinuclear EYFP-actin aggregates that colocalized with F-actin. During ATP depletion the G-actin fraction of EYFP-actin substantially decreased while endogenous and EYFP-F-actin increased. During intracellular ATP repletion, after 30 min of ATP depletion, there was a high degree of agreement between F-actin formation from EYFP-actin and endogenous actin. Our data indicate that EYFP-actin did not alter the characteristics of the endogenous actin cytoskeleton or the morphology of LLC-PK(10) cells. Furthermore, EYFP-actin is a suitable probe to study the spatial and temporal dynamics of actin cytoskeleton alterations in live proximal tubule cells during ATP depletion and ATP repletion.
Asunto(s)
Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Citoesqueleto/metabolismo , Túbulos Renales Proximales/metabolismo , Proteínas Luminiscentes/metabolismo , Actinas/genética , Animales , Proteínas Bacterianas/genética , Línea Celular , Citoesqueleto/ultraestructura , Immunoblotting , Túbulos Renales Proximales/citología , Proteínas Luminiscentes/genética , Microscopía Fluorescente , Faloidina/química , Faloidina/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Fibras de Estrés/metabolismo , PorcinosRESUMEN
UNLABELLED: The effect of hospitalization on an ESRD patient's hemoglobin (Hgb) level and erythropoietin (Epo) requirement has not been investigated. We postulated patients with end stage renal disease required an increased Epo dose to maintain stable Hgb during hospitalization and for a period following discharge. To evaluate this hypothesis, we conducted a retrospective chart review on 65 hemodialysis patients. All hemodialysis patients admitted for more than 2 days who did not have more than the index hospitalizations for 2 months prior to and following discharge were included. Multiple parameters including Hgb, Epo dose, intravenous iron dose, serum iron, TIBC, and ferritin during the 2 months before and the two months after hospitalization, Hgb at admission and discharge, Hgb trough, surgery, blood transfusions and co-morbid factors were evaluated. Statistical significance was evaluated using ANOVA or rank-sum testing, as appropriate. In 65 hemodialysis patients (24 M/41 F, age 58 +/- 2.2 years, mean +/- SEM), Hgb levels following discharge and for 2 subsequent months were significantly lower than 2 months prior to admission (11.4 +/- 0.25 vs. 10.7 +/- 0.22 g/dl, p < 0.01). This occurred in spite of an increase in Epo dose (128 +/- 14 vs. 185 +/- 21 U/kg/week, p < 0.0001) over this 2-month period. There was no difference in the iron saturation before and after hospitalization (22 vs. 23%,p > 0.05). There were also no apparent effects of comorbid factors, including surgery, or discharge diagnosis on the changes in Hgb or Epo requirements. However, patients who required a blood transfusion during the hospitalization had lower Hgb levels and higher Epo doses both prior to and after hospitalization, as well as lower Hgb trough levels. In addition, females had lower Hgb levels than males both prior to and after hospitalization, and were receiving a higher Epo dose 191 +/- 18 vs. 129 +/- 20 U/kg/week at 1 month and 215 +/- 18 U/kg/week vs. 134 +/- 22, p < 0.005 at 2 months after hospitalization. CONCLUSION: This study points out that hemodialysis patients experience a significant and prolonged decrease in Hgb levels after hospitalization, even despite a moderate increase in Epo dosing.
Asunto(s)
Anemia/tratamiento farmacológico , Eritropoyetina/administración & dosificación , Hospitalización/estadística & datos numéricos , Análisis de Varianza , Anemia/etiología , Transfusión Sanguínea , Femenino , Hemoglobinas/análisis , Humanos , Inflamación , Fallo Renal Crónico/complicaciones , Fallo Renal Crónico/terapia , Masculino , Persona de Mediana Edad , Diálisis Renal , Estudios Retrospectivos , Estadísticas no ParamétricasRESUMEN
Breakdown of proximal tubule cell apical membrane microvilli is an early-occurring hallmark of ischemic acute renal failure. Intracellular mechanisms responsible for these apical membrane changes remain unknown, but it is known that actin cytoskeleton alterations play a critical role in this cellular process. Our laboratory previously demonstrated that ischemia-induced cell injury resulted in dephosphorylation and activation of the actin-binding protein, actin depolymerizing factor [(ADF); Schwartz, N, Hosford M, Sandoval RM, Wagner MC, Atkinson SJ, Bamburg J, and Molitoris BA. Am J Physiol Renal Fluid Electrolyte Physiol 276: F544-F551, 1999]. Therefore, we postulated that ischemia-induced ADF relocalization from the cytoplasm to the apical microvillar microfilament core was an early event occurring before F-actin alterations. To directly investigate this hypothesis, we examined the intracellular localization of ADF in ischemic rat cortical tissues by immunofluorescence and quantified the concentration of ADF in brush-border membrane vesicles prepared from ischemic rat kidneys by using Western blot techniques. Within 5 min of the induction of ischemia, ADF relocalized to the apical membrane region. The length of ischemia correlated with the time-related increase in ADF in isolated brush-border membrane vesicles. Finally, depolymerization of microvillar F-actin to G-actin was documented by using colocalization studies for G- and F-actin. Collectively, these data indicate that ischemia induces ADF activation and relocalization to the apical domain before microvillar destruction. These data further suggest that ADF plays a critical role in microvillar microfilament destruction and apical membrane damage during ischemia.
Asunto(s)
Isquemia/metabolismo , Túbulos Renales Proximales/metabolismo , Proteínas de Microfilamentos/metabolismo , Factores Despolimerizantes de la Actina , Actinas/metabolismo , Animales , Destrina , Técnica del Anticuerpo Fluorescente , Túbulos Renales Proximales/irrigación sanguínea , Túbulos Renales Proximales/citología , Masculino , Membranas/metabolismo , Proteínas de Microfilamentos/orina , Microvellosidades/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
To study the intracellular mechanisms of aminoglycoside toxicity, we used a 1:1 fluorescent conjugate of Texas Red and gentamicin (TRG) to quantify early uptake dynamics in renal epithelial (LLC-PK(1)) cells. Utilizing a protocol that quenches TRG fluorescence from lysosomes, the bulk of intracellular accumulation, we determined a portion rapidly trafficked directly to the Golgi complex when identified by a FITC-conjugated lectin from Lens culinaris agglutinin (LCA). A kinetic study over 120 min on cells showing total and quenched TRG fluorescence was then carried out, and the fluorescence intensity from the images was quantified. Trafficking of TRG to the Golgi complex occurred within 15 min and accounted for approximately 20% of total cellular accumulation in the kinetic study. Colocalization studies using compartment-specific markers, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl sphingosine (C6-NBD ceramide) and LCA, for the TGN trans-Golgi network, and the cis/medial-Golgi compartments, respectively, determined colocalization occurred with both Golgi compartments. These data support the existence of a pathway that directly and rapidly shuttles a portion of internalized gentamicin to the Golgi complex. We believe this pathway may be responsible for the early negative effects seen on protein synthesis in renal proximal epithelia after aminoglycoside administration.
Asunto(s)
Células Epiteliales/metabolismo , Gentamicinas/farmacocinética , Aparato de Golgi/metabolismo , Riñón/metabolismo , Animales , Antibacterianos/química , Antibacterianos/farmacocinética , Transporte Biológico , Células Epiteliales/citología , Colorantes Fluorescentes/química , Gentamicinas/química , Aparato de Golgi/ultraestructura , Riñón/citología , Células LLC-PK1 , Porcinos , Xantenos/químicaRESUMEN
Acute renal failure (ARF) occurs frequently and results in an unacceptably high morbidity and mortality. There is no currently accepted specific therapy that alters the course of ischemic ARF. Recent experimental advances and continued funding of ARF studies should allow rapid progress in the new millennium. This will require novel approaches to both basic and clinical evaluations. New experimental models and studies evaluating multiple therapies are needed. In addition, methods to identify ARF early in its course are likely to improve outcomes. Clinical studies should employ very specific definitions of ARF, outcomes evaluated, indications for renal replacement therapy, and severity of illness evaluation methods. Such studies and aggressive preventative measures will significantly improve the incidence and outcome of ARF in the 21st century.
Asunto(s)
Lesión Renal Aguda/fisiopatología , Lesión Renal Aguda/terapia , Lesión Renal Aguda/mortalidad , Animales , Terapia Combinada/tendencias , Humanos , Isquemia/fisiopatología , Diálisis Renal , Terapia de Reemplazo Renal/tendenciasRESUMEN
Acute renal failure remains a common and life-threatening disease with a very high mortality. Renal replacement therapy only provides supportive care. The purpose of this review is to discuss the indications and complications of renal replacement therapies in acute renal failure. Various controversial issues such as biocompatibility of membranes, adequacy of dialysis and utilization of continuous renal replacement therapies in acute renal failure are also covered. The nutritional needs of a patient with acute renal failure receiving renal replacement therapy are also explained. Finally, the outcome of patients with acute renal failure requiring dialysis is discussed.
RESUMEN
The characteristic structure of polarized proximal tubule cells is drastically altered by the onset of ischemic acute renal failure. Distinctive apical brush border microvilli disruption occurs rapidly and in a duration-dependent fashion. Microvillar membranes internalize into the cytosol of the cell or are shed into the lumen as blebs. The microvillar actin core disassembles concurrent with or preceding these membrane changes. Actin and its associated binding proteins no longer interact to form these highly regulated apical membrane structures necessary for microvilli. The resultant epithelial cells have a reduced apical membrane surface that is not polarized either structurally, biochemically or physiologically. Furthermore, the changes in the apical microvilli result in tubular obstruction, reduced Na+ absorption, and partly explain the reduction in glomerular filtration rate. Recent evidence suggests these actin surface membrane alterations induced by ischemia are secondary to activation and relocation of the actin-associated protein, actin depolymerizing factor/cofilin, to the apical membrane domain. Activated (dephosphorylated) actin depolymerizing factor/cofilin proteins bind filamentous actin, increasing subunit treadmilling rates and filament severing. Once activated, the diffuse cytoplasmic distribution of the actin depolymerizing factor/cofilin protein relocalizes to the luminal membrane blebs. During recovery the actin depolymerizing factor/cofilin proteins are again phosphorylated and reassume their normal diffuse cytoplasmic localization. This evidence strongly supports the hypothesis that actin depolymerizing factor/cofilin proteins play a significant role in ischemia-induced injury in the proximal tubule cells.
Asunto(s)
Membrana Celular/fisiología , Isquemia/fisiopatología , Riñón/irrigación sanguínea , Riñón/fisiopatología , Animales , Células Epiteliales/fisiología , Epitelio/fisiopatología , Humanos , Riñón/fisiología , Microvellosidades/fisiología , Sodio/metabolismoAsunto(s)
Lesión Renal Aguda/metabolismo , Moléculas de Adhesión Celular/metabolismo , Isquemia/metabolismo , Riñón/metabolismo , Lesión Renal Aguda/etiología , Lesión Renal Aguda/fisiopatología , Humanos , Integrinas/metabolismo , Isquemia/complicaciones , Isquemia/fisiopatología , Riñón/irrigación sanguínea , Riñón/fisiopatología , Neutrófilos/metabolismo , Daño por Reperfusión/metabolismoRESUMEN
Epithelial cells mediate the unidirectional movement of selective compounds from one biological compartment to another. This is accomplished by having biochemically, structurally, and functionally distinctive apical and basolateral surface membrane domains separated by the cells' junctional complex. Derangement of this highly ordered situation can result in cell injury, dysfunction, and even death. For renal epithelial cells, both ischemia and polycystic kidney disease are known to result in a loss of surface membrane polarity. In both disease processes, this in turn plays an important role in cell and organ dysfunction.
Asunto(s)
Enfermedades Renales/patología , Riñón/citología , Riñón/patología , Animales , Células Epiteliales/fisiología , Salud , Humanos , Riñón/fisiología , Riñón/ultraestructura , Enfermedades Renales/fisiopatología , Urotelio/patologíaRESUMEN
Apical membrane of renal proximal tubule cells is extremely sensitive to ischemia, with structural alterations occurring within 5 min. These changes are felt secondary to actin cytoskeletal disruption, yet the mechanism responsible is unknown. Actin depolymerizing factor (ADF), a 19-kDa actin-binding protein, has recently been shown to play an important role in regulation of actin filament dynamics. Because ADF is known to mediate pH-dependent F-actin binding, depolymerization, and severing, and because ADF activation occurs by dephosphorylation, we questioned whether ADF played a role in microvilli microfilament disruption during ischemia. To test our hypothesis, we induced renal ischemia in the rat with the clamp model. Initial immunofluorescence and Western blot studies on cortical tissue documented the presence of ADF in proximal tubule cells. Under physiological conditions, ADF was distributed homogeneously throughout the cytoplasm, primarily in the Triton X-100-soluble fraction, and both phosphorylated (pADF) and nonphosphorylated forms were identified. During ischemia, marked alterations occurred. Intraluminal vesicle/bleb structures contained extremely high concentrations of ADF along with G-actin, but not F-actin. Western blot showed a rapidly occurring duration-dependent dephosphorylation of ADF. At 0-30 min of ischemia, total ADF levels were unchanged, whereas pADF decreased significantly to 72% and 19% of control levels, at 5 and 15 min, respectively. Urine collected under physiological conditions did not contain ADF or actin, whereas urine collected after 30 min of ischemia contained both ADF and actin. Reperfusion was associated with normalization of cellular pADF levels, pADF intracellular distribution, and repair of apical microvilli. These data suggest that activation of ADF during ischemia via dephosphorylation is, in part, responsible for apical actin disruption resulting in microvillar destruction and formation of intraluminal vesicles.
Asunto(s)
Isquemia/metabolismo , Túbulos Renales Proximales/metabolismo , Riñón/irrigación sanguínea , Proteínas de Microfilamentos/fisiología , Factores Despolimerizantes de la Actina , Actinas/metabolismo , Animales , Western Blotting , Destrina , Electroforesis en Gel Bidimensional , Técnica del Anticuerpo Fluorescente , Riñón/metabolismo , Masculino , Proteínas de Microfilamentos/metabolismo , Microscopía Confocal , Microvellosidades/metabolismo , Microvellosidades/ultraestructura , Fosforilación , Ratas , Ratas Sprague-DawleyRESUMEN
Acute renal failure remains an important clinical problem with little progress made in the therapeutic approach over the past 20-30 years. The purpose of this review is to discuss possible etiologies, their diagnosis, differentiation and possible prevention. The pathophysiology of prerenal azotemia and ischemic acute renal failure are also discussed. The importance of understanding different body volume components, the urine analysis and the FE Na(+) are explained.
RESUMEN
Significant advances have been made in understanding the pathophysiology of injury at the cellular level in ischemic acute renal failure. Alterations in the actin cytoskeleton are of central importance to the structural, physiological, and biochemical changes that occur in proximal tubule cells during acute ischemic injury. These cytoskeletal alterations occur rapidly and are dependent on the severity and duration of ischemic injury. Most importantly, alterations in the actin cytoskeleton are responsible for changes in the cell surface membrane that modify cell polarity, cell-cell interactions, and cell-matrix interactions. Ultimately, these cytoskeletal alterations play a major role in the decrement in glomerular filtration rate that is the hallmark of ischemic acute renal failure.
Asunto(s)
Lesión Renal Aguda/patología , Isquemia/patología , Actinas/análisis , Animales , Comunicación Celular , Polaridad Celular , Células Epiteliales/patología , Humanos , Riñón/irrigación sanguínea , Riñón/patologíaAsunto(s)
Lesión Renal Aguda/fisiopatología , Isquemia/fisiopatología , Lesión Renal Aguda/tratamiento farmacológico , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Sustancias de Crecimiento/farmacología , Sustancias de Crecimiento/fisiología , Proteínas de Choque Térmico/fisiología , Humanos , Isquemia/tratamiento farmacológico , Riñón/efectos de los fármacos , Riñón/fisiopatología , alfa-MSH/fisiología , alfa-MSH/uso terapéuticoRESUMEN
Aminoglycoside antibiotics are known to be internalized via endocytosis and have been associated with subcellular organelle dysfunction; however, the route of intracellular trafficking and their distribution remain largely unknown. To address these questions, a Texas Red conjugate of gentamicin (TRG) was synthesized for dual-labeling experiments with the endoplasmic reticulum, Golgi, and lysosomal markers DiOC6-3, C6-NBD-ceramide, and fluorescent dextrans, respectively. Confocal images were overlaid to determine areas of colocalization. Initial characterization studies of the fluorescent gentamicin analogue revealed that both internalization and accumulation were inhibited by excess unlabeled gentamicin. Furthermore, the fluorescent gentamicin label was colocalized with unlabeled gentamicin, using immunologic techniques. LLC-PK1 cells were exposed to the fluorescent gentamicin in media containing 1 mg/ml labeled gentamicin for 8 h and then either fixed or chased with gentamicin-free media for an additional 16 or 40 h (24 to 48 h total). Studies with fluorescent dextrans revealed rapid intracellular colocalization within the endosomal and lysosomal systems. Neither endoplasmic reticulum nor mitochondrial colocalization could be detected. However, Golgi colocalization was revealed using both confocal and electron microscopic techniques at 8 h of TRG incubation, and continued to be present for an additional 40 h. Protein synthetic rates were quantified and revealed decreased synthesis at the 24-h chase mark. These results suggest that TRG can serve as a fluorescent tracer for aminoglycoside trafficking within cells. The fluorescent marker remained associated with vesicular structures at all times and colocalized with the Golgi apparatus. It is postulated that this early association of gentamicin with the Golgi complex may be an avenue for delivery of aminoglycosides to other intracellular compartments.