ISOLATION OF PROXIMAL AND DISTAL TUBULE CELLS FROM HUMAN KIDNEY BY IMMUNOMAGNETIC SEPARATION

After collagenase digestion and Percoll density gradient centrifugatio n 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 monoc lonal antibody (mAb) against aminopeptidase M (APM, CD 13), specific o f the proximal tubule. Distal tubular (DT) cells were isolated through a mAb recognizing Tamm-Horsfall glycoprotein (THG), a specific antige n for the thick ascending limb and the early distal convoluted tubule. Cells of the proximal primary isolate were histochemically strongly p ositive for aminopeptidase M (98.6%), however, cells of the distal por tion were negative (98.7%). Ultrastructural analysis of PTC primary is olates revealed highly preserved brush border microvilli, well-develop ed endocytosis apparati and numerous mitochondria, whereas DTC primary isolates showed smaller cells with basolateral invaginations and less apical microvilli. Characterization by immunofluorescence indicated t he coexpression of cytokeratin and vimentin, whereas staining for desm in, smooth muscle actin, a fibroblast-specific marker and von Willebra nd factor was negative. Cultured PT and DT cells displayed different a denylate cyclase responsiveness to hormonal stimulation. PTH (10(-6) M ) increased cAMP production in distal cells up to 32.8-fold of the bas al level and in proximal only up to 3.5-fold (10(-8) M, DT 14.4X and P T 2.25X). Calcitonin stimulated adenylate cyclase in DT in a dose depe ndent fashion (10(-6)M, 4.3X; 10(-8)M, 2.25X), whereas only a low calc itonin 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 (o nly 1.3X the basal level). The data of this study indicate the proxima l and distal tubule origin of the cultured cells that were isolated ac cording to their segment-specific antigens.