INVESTIGADORES
GARRAMUÑO Patricia
artículos
Título:
A rapid enzymatic method for the isolation of defined kidney tubule fragments from mouse.
Autor/es:
CARSTEN A. WAGNER; ULRIKE LÜKEWILLE; PATRICIA GARRAMUÑO VALLÉS; SYLVIE BRETON; DENNIS BROWN; GERHARD H. GIEBISCH; JOHN P. GEIBEL
Revista:
PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY
Editorial:
Springer
Referencias:
Lugar: Germany; Año: 2003 vol. 446 p. 623 - 632
ISSN:
0031-6768
Resumen:
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.