Indirect immunoselection of late distal cell populations from rabbit kidney cortex.
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.
D000327:Adsorption; D000818:Animals; D000911:Antibodies, Monoclonal; D002458:Cell Fractionation; D002469:Cell Separation / Q000379:methods*; D002845:Chromatography / Q000379:methods; D005260:Female; D007158:Immunologic Techniques; D007672:Kidney Cortex / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D010101:Oxygen Consumption; D011817:Rabbits
D000911:Antibodies, Monoclonal