After collagenase digestion and Percoll density gradient centrifugation of human renal
tissue, tubular epithelial cells of the proximal and the distal segments were isolated
with an immunomagnetic method using MACS microbeads. To enrich proximal tubular (PT)
cells we used a monoclonal antibody (mAb) against aminopeptidase M (APM, CD 13), specific
of the proximal tubule. Distal tubular (DT) cells were isolated through a mAb recognizing
Tamm-Horsfall glycoprotein (THG), a specific antigen for the thick ascending limb
and the early distal convoluted tubule. Cells of the proximal primary isolate were
histochemically strongly positive for aminopeptidase M (98.6%), however, cells of
the distal portion were negative (98.7%). Ultrastructural analysis of PTC primary
isolates revealed highly preserved brush border microvilli, well-developed endocytosis
apparati and numerous mitochondria, whereas DTC primary isolates showed smaller cells
with basolateral invaginations and less apical microvilli. Characterization by immunofluorescence
indicated the coexpression of cytokeratin and vimentin, whereas staining for desmin,
smooth muscle actin, a fibroblast-specific marker and von Willebrand factor was negative.
Cultured PT and DT cells displayed different adenylate cyclase responsiveness to hormonal
stimulation. PTH (10(-6) M) increased cAMP production in distal cells up to 32.8-fold
of the basal level and in proximal only up to 3.5-fold (10(-8) M, DT 14.4x and PT
2.25x). Calcitonin stimulated adenylate cyclase in DT in a dose dependent fashion
(10(-6) M, 4.3x; 10(-8) M, 2.25x), whereas only a low calcitonin response was found
in PT cells (10(-6) M, 1.6x; 10(-8) M, 1.4x). AVP (10(-6) M) activated the distal
cAMP-production only up to 1.9x of the basal level, but the proximal cAMP-production
was negligible (only 1.3x the basal level). The data of this study indicate the proximal
and distal tubule origin of the cultured cells that were isolated according to their
segment-specific antigens.