Modelos experimentales en Nefrología. Lo que el nefrólogo debería conocer (1ª entrega)

La investigación en ciencias básicas es fundamental para el conocimiento de la patogénesis de las enfermedades y el posible desarrollo de nuevas estrategias terapéuticas. La representación necesaria para responder a los interrogantes que surgen del proceso de investigación requiere de la utilización de modelos experimentales En esta entrega abordamos el uso de los modelos in vivo en los estudios de función renal. Los esfuerzos que se han realizado para entender la fi siología humana se han apoyado extensamente en estos modelos. Los mamíferos, por su fi siología parecida a la de los humanos, y específi camente los roedores, han tenido un lugar de supremacía desde comienzos del siglo pasado, como modelo de enfermedades humanas. A partir de la década de los ´80 la facilidad de la manipulación genética de las células madre embrionarias en ratones dio origen a los modelos de ratones transgénicos. Los experimentos in vivo han sido esenciales para el conocimiento de la fi siología y la patología renal. La implementación de las técnicas de micropunción, junto al desarrollo de métodos para medir presiones hidrostáticas en pequeños compartimientos resultaron ser de importancia fundamental en los estudios de fi ltración glomerular, y sentaron las bases del conocimiento actual sobre este proceso ya sea en condiciones fi siológicas o patológicas.Fil: Monasterolo, Liliana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario; ArgentinaFil: Trumper, Laura. Universidad Nacional de Rosario; Argentin

Modelos experimentales en nefrología. Modelo de riñón aislado y perfundido

El modelo de riñón aislado y prefundido brinda la posibilidad de analizar la función renal bajo condiciones definidas y sin la intervención de factores sistémicos. Este modelo consiste en aislar al riñón de la vasculatura sistémica, y perfundirlo a través de la arteria renal con un medio de perfusión con características similares al plasma o a la sangre, conservando las características anatómicas, bioquímicas y funcionales del riñón intacto. Esto posibilita circunscribir factores extrarrenales que pueden complicar la interpretación de la información obtenida con experimentos realizados in vivo. Además, la utilización del órgano aislado permite el estudio de la función renal bajo condiciones en las que el operador puede modificar las variables a analizar de manera controlada. En el presente artículo se revisa la técnica de preparación, los criterios de viabilidad, las principales características hemodinámicas y tubulares, y las aplicaciones del modelo.The isolated perfused kidney model provides the ability to analyze the renal function under defined conditions without the intervention of systemic factors. In this model, the kidney is isolated from the systemic vasculature, and infused through the renal artery with a perfusion medium with the plasma or blood characteristics. The anatomical, biochemical and functional characteristics of the intact kidney is preserved. This allows to circumscribe extrarenal factors that can complicate the interpretation of the in vivo experiments. Moreover, the use of the isolated organ allows the study of renal function under conditions in which the operator can modify the analyzed variables in a controlled manner. In this article, the technique of preparation, viability criteria; and the main hemodynamic and tubular features and applications of the model are reviewed.Fil: Trumper, Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Monasterolo, Liliana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin

Effects of "in vivo" administration of baclofen on rat renal tubular function

The effects of the in vivo administration of baclofen on renal tubular transport and aquaporin-2 (AQP2) expression were evaluated. In conscious animals kept in metabolic cages, baclofen (0.01-1 mg/kg, s.c.) induced a dose-dependent increment in the urine flow rate (UFR) and in sodium and potassium excretion, associated with an increased osmolal clearance (Closm), a diminished urine to plasma osmolality ratio (Uosm/Posm) and a decrease in AQP2 expression. The above mentioned baclofen effects on functional parameters were corroborated by using conventional renal clearance techniques. Additionally, this model allowed the detection of a diminution in glucose reabsorption. Some experiments were performed with water-deprived or desmopressin-treated rats kept in metabolic cages. Either water deprivation or desmopressin treatment decreased the UFR and increased the Uosm/Posm. Baclofen did not change the Uosm/Posm or AQP2 expression in desmopressin-treated rats; but it increased the UFR and diminished the Uosm/Posm and AQP2 expression in water-deprived animals. These results indicate that in vivo administration of baclofen promotes alterations in proximal tubular transport, since glucose reabsorption was decreased. The distal tubular function was also affected. The increased Closm indicates an alteration in solute reabsorption at the ascending limb of the Henle's loop. The decreased Uosm/Posm and AQP2 expression in controls and in water-deprived, but not in desmopressin-treated rats, lead us to speculate that some effect of baclofen on endogenous vasopressin availability could be responsible for the impaired urine concentrating ability, more than any disturbance in the responsiveness of the renal cells to the hormone.Fil: Donato, Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Pisani, Gerardo Bruno. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Trumper, Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Monasterolo, Liliana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Are all edges equal? Microclimatic conditions, geographical orientation and biological implications in a fragmented forest

In fragmented forests the edges experience changes in microclimatic conditions, which are referred to as “abiotic edge effect”, and differ according to geographical orientation and season. These microclimatic changes could influence the development rate of the organisms (particularly for movement - restricted ones like leaf miner larvae and their parasitoids)and, consequently, their population dynamics. The aim of this study was to compare the abiotic edge effect in differently oriented edges in summer and winter seasons, and evaluate possible implications on insect development, using a simulation based on a degree-day approach. To compare the abiotic edge effect, we took continuous and point measurements of microclimatic variables such as temperature, humidity, wind velocity and interception of photosynthetically active radiation in three microhabitats: interior (I), north-facing edges (NE)and south-facing edges (SE)of six fragments of Chaco Serrano forest, in winter and summer seasons. As we expected, the microclimatic edge effect was greater in NE compared to SE in both seasons. In winter, the differences were more pronounced only for continuous measurements of temperature. Our simulation exercise suggested that differences in temperature among microhabitats may have biological implications on insect populations, affecting their development time. At larger scales, an increase of temperature could lead to more frequent pest outbreaks which could extend their distribution range to higher latitudes. Our findings emphasize the need to consider geographical orientation and season variations when studying edge effects on insect populations. In the context of climate change, organisms could restrict their distributions to habitats with more suitable conditions, becoming microrefuges that could allow them to survive. Moreover, in fragmented landscapes, identifying microhabitats benefitting or harming insect populations is an important step to design management strategies towards pest control or natural enemy conservation in agricultural settings.Fil: Bernaschini, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Trumper, Eduard. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuaria Manfredi; ArgentinaFil: Valladares, Graciela Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Salvo, Silvia Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentin

Acidosis-induced downregulation of hepatocyte mitochondrial aquaporin-8 and ureagenesis from ammonia

It has been proposed that during metabolic acidosis, the liver downregulates mitochondrial ammonia detoxification via ureagenesis, a bicarbonate-consuming process. Since we previously demonstrated that hepatocyte mitochondrial aquaporin-8 channels (mtAQP8) facilitate the uptake of ammonia and its metabolism into urea, we studied whether mtAQP8 is involved in the liver adaptive response to acidosis. Primary cultured rat hepatocytes were adapted to acidosis by exposing them to culture medium at pH 7.0 for 40 h. Control cells were exposed to pH 7.4. Hepatocytes exposed to acid medium showed a decrease in mtAQP8 protein expression (-30%, p<0.05). Ureagenesis from ammonia was assessed by incubating the cells with 15N-labeled ammonia and measuring 15N-labeled urea synthesis by nuclear magnetic resonance. Reduced ureagenesis was found in acidified hepatocytes (-31%, p<0.05). In vivo studies in rats subjected to 7-days acidosis also showed decreased protein expression of hepatic mtAQP8 (-50%, p<0.05) and reduced liver urea content (-35%; p<0.05). In conclusion, our in vitro and in vivo data suggest that hepatic mtAQP8 expression is downregulated in acidosis, a mechanism that may contribute to decreased ureagenesis from ammonia in response to acidosis.Fil: Molinas, Sara Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Soria, Leandro Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Marrone, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Danielli, Mauro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); ArgentinaFil: Trumper, Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Marinelli, Raul Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Fisiología Experimental (i); Argentin

Glutamine protection in an experimental model of acetaminophen nephrotoxicity

Acetaminophen (APAP) is a widely prescribed analgesic and antipyretic drug. In the present work, we studied the effects of glutamine (Gln) in an in vivo model of APAP-induced nephrotoxicity in male Wistar rats. Renal function, histological characteristics, and Na+,K+-ATPase cortical abundance and distribution were analyzed. The appearance of HSP70 and actin in urine was also evaluated. Myeloperoxidase (MPO) activity in cortical tissue was measured as an index of the inflammatory response. Gln administration 30 min before APAP protected from the renal functional and histological damage promoted by APAP. Rats that received the dual treatment Gln and APAP (Gln/APAP) showed the same level of Na+,K+-ATPase cortical induction as APAP-treated animals, but the enzyme maintained its normal basolateral localization. HSP70 abundance was increased up to the same level in the Gln, APAP, and Gln/APAP groups. Urinary HSP70 and actin were detected only in the APAP-treated animals, reinforcing the protection of renal tubular integrity afforded by the Gln pretreatment. Gln pretreatment also protected from the increment in MPO activity promoted by APAP. Our results support the idea that Gln pretreatment could be a therapeutic option to prevent APAP-induced renal injury.Fil: Brovedan, Marco Alcides. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Area Farmacología; ArgentinaFil: Molinas, Sara Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Area Farmacología; ArgentinaFil: Pisani, Gerardo Daniel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Ciencias Fisiológicas. Área Morfología; ArgentinaFil: Monasterolo, Liliana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Area Farmacología; ArgentinaFil: Trumper, Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Area Farmacología; Argentina. Universidad Nacional de Rosario. Centro de Investigaciones de la Universidad Nacional de Rosario; Argentin

Localization and functional activity of cytochrome P450 side chain cleavage enzyme (CYP11A1) in the adult rat kidney

Cumulative evidence demonstrated effective downstream metabolism of pregnenolone in renal tissue. The aim of this study was to evaluate the expression and functional activity of cytochrome P450 side chain cleavage enzyme (CYP11A1), which converts cholesterol into pregnenolone, in adult rat kidney. Immunohistochemical labeling for CYP11A1 was observed in renal cortex and medulla, on structures identified as distal convoluted tubule and thick ascending limb of Henle's loop, respectively. Immunoblotting analysis corroborated the renal expression of the protein in inner mitochondrial membrane fractions. The incubation of isolated mitochondria with the membrane-permeant cholesterol analogue 22R-hydroxycholesterol resulted in efficient formation of pregnenolone, the immediate precursor for the synthesis of all the steroid hormones. The low progesterone production rate observed in these experiments suggested a poor activity of 3β-hydroxysteroid dehydrogenase enzyme in renal mitochondria. The steroidogenic acute regulatory protein (StAR), involved in the mitochondrial import of cholesterol, was detected in renal tissue at both mRNA and protein level. Immunostaining for StAR showed similar distribution to that observed for CYP11A1. The expression of StAR and CYP11A1 was found to be higher in medulla than in cortex. This enhanced expression of steroidogenesis-related proteins correlated with a greater pregnenolone synthesis rate and higher steroid hormones tissular content measured in medulla. In conclusion, we have established the expression and localization of StAR and CYP11A1 protein, the ability of synthesizing pregnenolone and a region-specific content of sex hormones in the adult rat kidney. These data clearly show that the kidney is a steroid hormones synthesizing organ. It is proposed that the existence in the kidney of complete steroidogenic machinery would respond to a physiological significance. © 2010 Elsevier Ireland Ltd.Fil: Pagotto, Melina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Roldán, María Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Pagotto, Romina María del Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Lugano, Maria C.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Pisani, Gerardo. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Rogic, Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Molinas, Sara Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; ArgentinaFil: Trumper, Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Pignataro, Omar Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Monasterolo, Liliana Alicia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin