AJP - Renal Physiology, Vol 250, Issue 3 386-F395, Copyright © 1986 by American Physiological Society
Indirect immunoselection of late distal cell populations from rabbit kidney cortex
A. Vandewalle, M. Tauc, F. Cluzeaud, P. Ronco, F. Chatelet, P. Verroust and P. Poujeol
This study describes a method for the separation of distal cell populations
based on the sequestration of proximal cells on immunoadsorbent columns
(CNBr-activated Sepharose 6MB) bound with three brush-border monoclonal
antibodies (S6-Mab). A high yield of isolated cell suspension from rabbit
kidney cortex was prepared by mechanical dissociation after perfusion and
incubation of the kidneys with 10(-3) M EDTA. The sequestration of the
proximal cells was achieved in two sequential chromatographic steps. About
92% of the applied cells were first retained on an S6-Mab column after a
60-min stationary stage and the unbound cells were submitted by direct flow
to a second S6-Mab column. In such conditions, 8 X 10(6) cells were
recovered when starting with 331 X 10(6) cortical cells. The efficiency of
the proximal cell depletion process was confirmed by an 80% decrease in
brush-border enzymes, a very low phosphoenolpyruvate carboxykinase
activity, and absence of cells bearing long microvilli, as ascertained by
electron microscopy. This immunodepleted cell population presented the
enzymatical characteristics of cells from the more distal segments. As
compared with the initial cell suspension, these cells exhibited higher
hexokinase (2.3 times), succinate dehydrogenase (1.5 times), and
Na+-K+-ATPase (2.6 times) activities. In addition, adenylate cyclase
activities remained sensitive to parathormone, arginine vasopressin, and
isoproterenol. The functional capacity of these immunodepleted cells was
assessed by an almost complete exclusion of eosin dye, a low Na+ and high
K+ intracellular content, and a high respiratory rate of oxygen
consumption. In conclusion, this immunoselective process makes it possible
to obtain subpopulations of renal cortical cells possessing the main
characteristics of the distal, connecting, and collecting cells for
physiological and metabolic studies.
