Use of cultured tubular cells isolated from human urine for investigation of renal transporter

Aim:To facilitate the understanding of the transporter function of human re nal tubular cells, we have developed a simple method using primary cultured proximal tubule (PT) cells isolated from voided urine. Methods: PT cells g rown to confluence on glass coverslips could be identified by parallel arra ys of spindle cells and hemicyst formation. Brush-border gamma-glutamyl tra nspeptidase (gamma GTP) activity was histochemically identified. Apical mem brane Na+/H+ exchanger (NHE) activity was measured by monitoring changes in intracellular pH (pH(i)) after an acid load in a single cell level using t he pH-sensitive dye 2'7'-bis-(2-carboxyethyl)-5.6'carboxyfluorescein (BCECF ). Results: Amiloride and 5-(N-ethyl-N-isopropyl) amiloride (EIPA) inhibite d the NHE activity with half-maximal inhibition values (IC50) of 15.3 and 4 .0 mu M, respectively. NHE-3 mRNA was detected by the RT-PCR technique in c lonally proliferated PT cells. Conclusion: These results suggest that cultu red PT cells isolated from human urine express amiloride-resistant NHE-3 ac tivity on the apical membranes, which can be compared to functional propert ies of PT in vivo. Our experimental strategy offers a useful experimental a pproach to investigating human renal tubular transport function in vitro.