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

• Published in: Kidney international Vol. 59; no. 2; pp. 554 - 564
• Main Authors: Helbert, Mark J.F., Dauwe, Simonne E.H., De Broe, Marc E.
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01-02-2001; Nature Publishing; Elsevier Limited
• Subjects: Animal cells; Biological and medical sciences; cell culture; Cell cultures. Hybridization. Fusion; Cell Separation - methods; epithelial membrane antigen; Flow Cytometry; fluorescence activated cell sorting; Fundamental and applied biological sciences. Psychology; Humans; Immunologic Techniques; Kidney Cortex; Kidney Tubules, Collecting - cytology; Kidney Tubules, Collecting - metabolism; Kidney Tubules, Distal - cytology; Kidney Tubules, Distal - metabolism; L1-cell adhesion molecule; Leukocyte L1 Antigen Complex; markers to isolate cells; Membrane Glycoproteins - metabolism; Molecular and cellular biology; Mucin-1 - metabolism; Nephrons - metabolism; Neural Cell Adhesion Molecules - metabolism; tubular cell pathology; markers to isolate cells; epithelial membrane antigen; tubular cell pathology; cell culture; L1-cell adhesion molecule; fluorescence activated cell sorting; Activated state; Human; Cell culture; Flow cytometry; Renal tubule; Distal; Cell adhesion molecule; Biological marker; Collecting duct; Immunofluorescence; Cell
• ISSN: 0085-2538; 1523-1755
• DOI: 10.1046/j.1523-1755.2001.059002554.x

Flow cytometric immunodissection of the human distal tubule and cortical collecting duct system.
In recent years, considerable efforts were drawn to isolate human distal tubule (DT) and collecting duct (CD) cells with more or less success. Here, we present a procedure for isolating human DT cells [thick ascending limb (TAL)/distal convoluted tubule (DCT)] and CD system cells (connecting tubule/initial CD) as separate populations within the same kidney specimen, applying monoclonal antibodies in fluorescence-activated cell sorting (FACS) and culturing them.
We tested antibodies directed against the DT/CD system antigens, epithelial membrane antigen (EMA) and L1-cell adhesion molecule (L1-CAM). Segmental and subsegmental expressions were first assessed by using morphologic and histotopographic criteria, and by comparing sections with adjacent sections stained for expression of well-defined distal subsegment-specific markers. Immunoreactive cells were further characterized by dual immunostaining using cell type-specific markers. As a second step, cells obtained by collagenase digestion of normal renal cortical tissue were flow sorted following labeling with aforementioned antibodies and cultured.
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 decreased 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 system cells as a heterogeneous mixture. Flow sorting of only the most strongly EMA-positive cells allowed purification of DT cells only, mainly TAL cells 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/cortical CD). Primary cultures of DT and CD system cells rapidly developed into confluent monolayers, and retained antigenic and functional properties inherent to their segments of origin.
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.