Mjf. Helbert et al., Flow cytometric immunodissection of the human distal tubule and cortical collecting duct system, KIDNEY INT, 59(2), 2001, pp. 554-564
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.