TRANSPORT CHARACTERISTICS OF HUMAN PROXIMAL TUBULE CELLS IN PRIMARY CULTURE

In order to establish an in vitro model for studying human proximal tu bule transport, primary culture of human proximal tubule cells (PTC) w as carried out using an improved technique and the properties of these cells were characterised in detail. Using a combination of collagenas e treatment, mechanical sieving and isopycnic ultracentrifugation, lar ge numbers of highly purified populations of PTC were isolated and pro pagated from histologically normal regions of human nephrectomy specim ens. Cultured human PTC demonstrated typical histologic and ultrastruc tural 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 diffusio n of inulin, was achieved on porous membrane inserts within 6-8 days. Confluent monolayers generated Na+, K+, Cl-, HCO3- and PO43- concentra tion gradients between apical and basolateral medium compartments, whi ch correlated well with the reabsorption processes known to occur in h uman PTC in vivo. A number of polarised transport systems were demonst rated, including phlorizin-inhibitable apical Na+-glucose transport, P TH-inhibitable apical Na+-phosphate transport, probenecid-inhibitable organic anion transport and quinine-inhibitable organic cation transpo rt. Using microspectrofluorimetric and Na-22(+) uptake measurements, p harmacologically distinct apical and basolateral sodium-hydrogen excha ngers (NHE) were identified. Apical NHE was significantly inhibited by micromolar concentrations of phorbol esters, ethylisopropylamiloride (EIPA) and 3-methylsulphonyl-4-piperidinobenzoylguanidine methanesulph onate (HOE694). The mean resting intracellular pH of human PTC was 7.2 3 +/- 0.04 and the mean intrinsic buffering capacity following a 20 mm ol/L NH4Cl prepulse was 28.45 +/- 0.96 mmol/l/unit pH. The results sug gest that human PTC, prepared for culture as described herein, maintai n morphological and physiological properties characteristic of the seg ment in vivo. The method therefore provides a useful model for the stu dy of highly polarised transport processes in the human proximal tubul e.