Isolation of proximal and distal tubule cells from human kidney by immunomagnetic separation

Abstract

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 proxireal 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%). Ultrastrustural 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.