Vasopressin receptor-mediated functional signaling pathway in primary cilia of renal epithelial cells

The primary cilium of renal epithelial cells is a nonmotile sensory organelle, implicated in mechanosensory transduction signals. Recent studies from our laboratory indicate that renal epithelial primary cilia display abundant channel activity; however, the presence and functional role of specific membrane receptors in this organelle are heretofore unknown. Here, we determined a functional signaling pathway associated with the type 2 vasopressin receptor (V2R) in primary cilia of renal epithelial cells. Besides their normal localization on basolateral membrane, V2R was expressed in primary cilia of LLC-PK1 renal epithelial cells. The presence of V2R in primary cilia was determined by spontaneous fluorescence of a V2R-gfp chimera and confirmed by immunocytochemical analysis of wild-type LLC-PK1 cells stained with anti-V2R antibodies and in LLC-PK1 cells overexpressing the V2R-Flag, with anti-Flag antibody. Ciliary V2R colocalized with adenylyl cyclase (AC) type V/VI in all cell types tested. Functional coupling of the receptors with AC was confirmed by measurement of cAMP production in isolated cilia and by testing AVP-induced cation-selective channel activity either in reconstituted lipid bilayers or subjected to membrane-attached patch clamping. Addition of either 10 μM AVP (trans) or forskolin (cis) in the presence but not the absence of ATP (1 mM, cis) stimulated cation-selective channel activity in ciliary membranes. This channel activity was reduced by addition of the PKA inhibitor PKI. The data provide the first demonstration for the presence of V2R in primary cilia of renal epithelial cells, and a functional cAMP-signaling pathway, which targets ciliary channel function and may help control the sensory function of the primary cilium.Fil: Raychowdhury, Malay K.. Massachusetts General Hospital; Estados Unidos. Harvard Medical School; Estados UnidosFil: Ramos, Arnolt J.. Massachusetts General Hospital; Estados Unidos. Harvard Medical School; Estados UnidosFil: Zhang, Peng. Massachusetts General Hospital; Estados Unidos. Harvard Medical School; Estados UnidosFil: McLaughin, Margaret. Harvard Medical School; Estados Unidos. Massachusetts General Hospital; Estados UnidosFil: Dai, Xiao-Qing. University of Alberta; CanadáFil: Chen, Xing-Zhen. University of Alberta; CanadáFil: Montalbetti, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; ArgentinaFil: Cantero, Maria del Rocio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; ArgentinaFil: Ausiello, Dennis A.. Massachusetts General Hospital; Estados Unidos. Harvard Medical School; Estados UnidosFil: Cantiello, Horacio Fabio. Massachusetts General Hospital; Estados Unidos. Harvard Medical School; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; Argentin

Synchronization modulation increases transepithelial potentials in MDCK monolayers through Na/K pumps

Peer reviewedPublisher PD

Posible efecto protector renal de Solanum sisymbriifolium LAM. (SOLANACEAE) en ratas hipertensas por Nω-Nitro-L-Arginina metilester.

En este trabajo se propone evaluar la potencial actividad protectora renal del extracto bruto de la raíz de S. sisymbriifolium en ratas con hipertensión inducida por el Nω-Nitro-L-arginina metilester (L-NAME).CONACYT - Consejo Nacional de Ciencias y TecnologíaPROCIENCI

Toxicidad sub crónica y actividad analgésica in vivo del extracto clorofórmico de las hojas de Calea urticifolia (Juanislama

Introduction: The population uses medicinal plants indiscriminately to treat diseases, with the believe that they are safe and lack adverse effects. Objective: To determine the in vivo toxicological and analgesic effect of the chloroform extract of Calea urticifolia leaves. Methodology: The toxicological study was performed using a 90- day sub-chronic toxicity test in NIH mice, at repeated and continuous doses. . Blood biochemistry, hematology and histopathological examination of organs were performed. The analgesic activity was evaluated in vivo using a model of abdominal contortions. Results: The administration of the plant extract caused the appearance of clinical signs of toxicity, alterations in hematic parameters and blood biochemistry, as well as histological alterations in some of organs. The analgesic activity at 100 mg/kg was similar to the Indomethacin drug. Conclusion: Despite the proven analgesic activity, according to the observed toxicological effects in this study, the prolonged use of Calea urticifolia leaves is not recommended for the treatment of diseasesIntroducción: La población utiliza la medicina a base de hierbas de forma indiscriminada basándose en la creencia de que las plantas medicinales carecen de efectos adversos. Objetivo: Determinar in vivo el efecto toxicológico y analgésico del extracto clorofórmico de las hojas de Calea urticifolia. Metodología: El estudio toxicológico fue realizado mediante la prueba de toxicidad subcrónica de 90 días, a dosis repetidas y continuas en ratones NIH. Se realizaron análisis de bioquímica sanguínea, hematología y el examen histopatológico de órganos. La actividad analgésica fue evaluada con el modelo in vivo de contorsiones abdominales. Resultados: La administración del extracto vegetal provocó la aparición de signos clínicos de toxicidad, alteraciones en los parámetros hematólogos y bioquímica sanguínea, además alteraciones histológicas en algunos de los órganos. La actividad analgésica a 100 mg/kg resultó comparable con el fármaco indometacina. Conclusión: Pese a la actividad analgésica demostrada, y de acuerdo a los efectos toxicológicos encontrados, no se recomienda el uso prolongado de las hojas de Calea urticifolia, para el tratamiento de enfermedade

Enzyme-Catalyzed Macrocyclization of Long Unprotected Peptides

A glutathione S-transferase (GST) catalyzed macrocyclization reaction for peptides up to 40 amino acids in length is reported. GST catalyzes the selective SNAr reaction between an N-terminal glutathione (GSH, γ-Glu-Cys-Gly) tag and a C-terminal perfluoroaryl-modified cysteine on the same polypeptide chain. Cyclic peptides ranging from 9 to 24 residues were quantitatively produced within 2 h in aqueous pH = 8 buffer at room temperature. The reaction was highly selective for cyclization at the GSH tag, enabling the combination of GST-catalyzed ligation with native chemical ligation to generate a large 40-residue peptide macrocycle.Massachusetts Institute of Technology (MIT startup funds)National Institutes of Health (U.S.) (grant GM101762)Damon Runyon Cancer Research Foundation (Award)Sontag Foundation (Distinguished Scientist Award)Amgen Inc. (Summer Graduate Research Fellowship

Intracellular iron uptake is favored in Hfe-KO mouse primary chondrocytes mimicking an osteoarthritis-related phenotype

HFE-hemochromatosis is a disease characterized by a systemic iron overload phenotype mainly associated with mutations in the HFE protein (HFE) gene. Osteoarthritis (OA) has been reported as one of the most prevalent complications in HFE-hemochromatosis patients, but the mechanisms associated with its onset and progression remain incompletely understood. In this study, we have characterized the response to high iron concentrations of a primary culture of articular chondrocytes isolated from newborn Hfe-KO mice and compared the results with that of a similar experiment developed in cells from C57BL/6 wild-type (wt) mice. Our data provide evidence that both wt- and Hfe-KO-derived chondrocytes, when exposed to 50 mu M iron, develop characteristics of an OA-related phenotype, such as an increased expression of metalloproteases, a decreased extracellular matrix production, and a lower expression level of aggrecan. In addition, Hfe-KO cells also showed an increased expression of iron metabolism markers and MMP3, indicating an increased susceptibility to intracellular iron accumulation and higher levels of chondrocyte catabolism. Accordingly, upon treatment with 50 mu M iron, these chondrocytes were found to preferentially differentiate toward hypertrophy with increased expression of collagen I and transferrin and downregulation of SRY (sex-determining region Y)-box containing gene 9 (Sox9). In conclusion, high iron exposure can compromise chondrocyte metabolism, which, when simultaneously affected by an Hfe loss of function, appears to be more susceptible to the establishment of an OA-related phenotype.European Regional Development FundEuropean Union (EU) [EMBRC.PT Alg-01-0145-FEDER-022121, Norte-01-0145-FEDER-000012]Fundacao para a Ciencia e a TecnologiaPortuguese Foundation for Science and Technology [SFRH/BD/77056/2011]Portuguese Foundation for Science and TechnologyPortuguese Foundation for Science and TechnologyPortuguese Science and Technology FoundationPortuguese Foundation for Science and Technologyinfo:eu-repo/semantics/publishedVersio