Flow cytometric immunodissection of the human distal tubule and cortical collecting duct system

Background. In recent years, considerable efforts were drawn to isolate hum an distal tubule (DT) and collecting duct (CD) cells with more or less succ ess. Here, we present a procedure for isolating human DT cells [thick ascen ding limb (TAL)/ distal convoluted tubule (DCT)] and CD system cells (conne cting tubule/initial CD) as separate populations within the same kidney spe cimen, applying monoclonal antibodies in fluorescence-activated cell sortin g (FACS) and culturing them. Methods. We tested antibodies directed against the DT/CD system antigens. e pithelial membrane antigen (EMA) and L1-cell adhesion molecule (L1-CAM). Se gmental and subsegmental expressions were first assessed by using morpholog ic and histotopographic criteria, and by comparing sections with adjacent s ections stained for expression of well-defined distal subsegment-specific m arkers. Immunoreactive cells were further characterized by dual immunostain ing using cell type-specific markers. As a second step, cells obtained by c ollagenase digestion of normal renal cortical tissue were flow sorted follo wing labeling with aforementioned antibodies and cultured. Results. EMA expression was found on all cells present in the DT and in the CD system. Its expression was most abundant in TAL and from thereon decrea sed gradually along the course of the DT and CD system. Flow sorting of all EMA-expressing cells resulted in identification/isolation of DT and CD sys tem cells as a heterogeneous mixture. Flow sorting of only the most strongl y EMA-positive cells allowed purification of DT cells only, mainly TAL cell s as shown by Tamm-Horsfall protein expression on >80% of sorted cells. L1- CAM was expressed in only the CD system, and sorting of all L1-CAM-positive cells allowed >95% purification of CD system cells (connecting tubule/cort ical CD). Primary cultures of DT and CD system cells rapidly developed into confluent monolayers, and retained antigenic and functional properties inh erent to their segments of origin. Conclusion. Our study presents a procedure for isolating and culturing pure populations of human DT cells and CD system cells as separate populations, using antibodies to the best available markers in FACS.