Transport characteristics of human proximal tubule cells in primary culture

Summary: In order to establish an in vitro model for studying human proximal tubule transport, primary culture of human proximal tubule cells (PTC) was carried out using an improved technique and the properties of these cells were characterised in detail. Using a combination of collagenase treatment, mechanical sieving and isopycnic ultracentrifugation, large numbers of highly purified populations of PTC were isolated and propagated from histologically normal regions of human nephrectomy specimens. Cultured human PTC demonstrated typical histologic and ultrastructural morphologies, well-preserved brush border enzyme activities, and cyclic adenosine monophosphate (cAMP) production which was stimulated by parathyroid hormone (PTH) but not by vasopressin. Tight confluence, as evidenced by relative impermeability to the paracellular diffusion of inulin, was achieved on porous membrane inserts within 6–8 days. Confluent monolayers generated Na+, K+, Cl−, HCO3− and PO43- concentration gradients between apical and basolateral medium compartments, which correlated well with the reabsorption processes known to occur in human PTC in vivo. A number of polarised transport systems were demonstrated, including phlorizin-inhibitable apical Na+-glucose transport, PTH-inhibitable apical Na+-phosphate transport, probenecid-inhibitable organic anion transport and quinine-inhibitable organic cation transport. Using microspectrofluorimetric and 22Na+ uptake measurements, pharmacologically distinct apical and basolateral sodium-hydrogen exchangers (NHE) were identified. Apical NHE was significantly inhibited by micromolar concentrations of phorbol esters, ethylisopropylamiloride (EIPA) and 3-methylsulphonyl-4-piperidino-benzoylguanidine methanesulphonate (HOE694). the mean resting intracellular pH of human PTC was 7.23 ± 0.04 and the mean intrinsic buffering capacity following a 20 mmol/L NH4Cl prepulse was 28.45 ± 0.96 mmol/L/unit pH. the results suggest that human PTC, prepared for culture as described herein, maintain morphological and physiological properties characteristic of the segment in vivo. the method therefore provides a useful model for the study of highly polarised transport processes in the human proximal tubule.