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dc.contributor.author
Wagner, Carsten A.  
dc.contributor.author
Lükewille, Ulrike  
dc.contributor.author
Garramuño, Patricia  
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Breton, Sylvie  
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Brown, Dennis  
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Giebisch, Gerhard H.  
dc.contributor.author
Geibel, John P.  
dc.date.available
2021-03-10T20:52:19Z  
dc.date.issued
2003-08  
dc.identifier.citation
Wagner, Carsten A.; Lükewille, Ulrike; Garramuño, Patricia; Breton, Sylvie; Brown, Dennis; et al.; A rapid enzymatic method for the isolation of defined kidney tubule fragments from mouse; Springer; Pflugers Archiv-European Journal of Physiology; 446; 5; 8-2003; 623-632  
dc.identifier.issn
0031-6768  
dc.identifier.uri
http://hdl.handle.net/11336/128018  
dc.description.abstract
The increasing number of available genetically manipulated mice makes it necessary to develop tools and techniques for examining the phenotypes of these animals. We have developed a straightforward and rapid method for the isolation of large quantities of single tubule fragments from the mouse kidney. Immunohistochemistry, electron microscopy, and fluorescence microscopy were used to evaluate the viability, functional characteristics, and morphology of proximal tubules (PT), and collecting ducts from cortex (CCD) and inner stripe of the outer medulla (ISOMCD). Tubules were isolated using a modified collagenase digestion technique, and selected under light microscopy for experimentation. Electron microscopy and trypan blue exclusion showed that a large portion of unselected proximal tubules were damaged by the digestion procedure. The selected tubules, however, all excluded trypan blue, indicating that the plasma membrane had remained intact. Immunocytochemistry on isolated CCD showed normal distribution of H+-ATPase, pendrin, and anion exchanger-1 (AE-1) staining. The pH-sensitive dye 2′,7′ -bis(2-carboxylethyl)-5(6)-carboxyfluorescein (BCECF) was used to measure Na+-dependent and -independent intracellular pH (pHi) recovery rates in PT, and in single intercalated cells of CCD and ISOMCD fragments. Na+-dependent pHi-recovery was 0.144±0.008 (PT), 0.182±0.013 (CCD), and 0.112±0.010 pH units/min. (ISOMCD). Na+-independent pHi recovery was found in all three segments (PT: 0.021±0.002, CCD: 0.037±0.002, ISOMCD: 0.033± 0.002 pH units/min) and was sensitive to concanamycin. In summary, we have developed a new technique for rapid and straightforward preparation of large quantities of defined tubule fragments from mouse kidney. Using this technique, the first measurements of plasma membrane vacuolar H +-ATPase activities in mouse PT and collecting duct were made. This technique will facilitate further characterization of kidney function in normal and genetically manipulated animals.  
dc.format
application/pdf  
dc.language.iso
eng  
dc.publisher
Springer  
dc.rights
info:eu-repo/semantics/restrictedAccess  
dc.rights.uri
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/  
dc.subject
COLLECTING DUCT  
dc.subject
ISOLATED RENAL TUBULES  
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MOUSE KIDNEY  
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PH MEASUREMENT  
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VACUOLAR H +-ATPASE  
dc.subject.classification
Urología y Nefrología  
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Medicina Clínica  
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CIENCIAS MÉDICAS Y DE LA SALUD  
dc.title
A rapid enzymatic method for the isolation of defined kidney tubule fragments from mouse  
dc.type
info:eu-repo/semantics/article  
dc.type
info:ar-repo/semantics/artículo  
dc.type
info:eu-repo/semantics/publishedVersion  
dc.date.updated
2021-01-18T15:48:14Z  
dc.journal.volume
446  
dc.journal.number
5  
dc.journal.pagination
623-632  
dc.journal.pais
Alemania  
dc.journal.ciudad
Germany  
dc.description.fil
Fil: Wagner, Carsten A.. Universitat Zurich; Suiza. University of Yale; Estados Unidos  
dc.description.fil
Fil: Lükewille, Ulrike. University of Yale; Estados Unidos  
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Fil: Garramuño, Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; Argentina  
dc.description.fil
Fil: Breton, Sylvie. Massachusetts General Hospital; Estados Unidos  
dc.description.fil
Fil: Brown, Dennis. Massachusetts General Hospital; Estados Unidos  
dc.description.fil
Fil: Giebisch, Gerhard H.. University of Yale; Estados Unidos  
dc.description.fil
Fil: Geibel, John P.. University of Yale; Estados Unidos  
dc.journal.title
Pflugers Archiv-European Journal of Physiology  
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/url/https://link.springer.com/article/10.1007/s00424-003-1082-3  
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/doi/http://dx.doi.org/10.1007/s00424-003-1082-3