Use of cultured tubular cells isolated from human urine for investigation of renal transporter
Inoue, C.N.; Kondo, Y.; Ohnuma, S.; Morimoto, T.; Nishio, T.; Iinuma, K.
Clinical Nephrology 53(2): 90-98
2000
ISSN/ISBN: 0301-0430 PMID: 10711410 Document Number: 513275
Aim: To facilitate the understanding of the transporter function of human renal tubular cells, we have developed a simple method using primary cultured proximal tubule (PT) cells isolated from voided urine. Methods: PT cells grown to confluence on glass coverslips could be identified by parallel arrays of spindle cells and hemicyst formation. Brush-border gamma-glutamyl transpeptidase (gammaGTP) activity was histochemically identified. Apical membrane Na+/H+ exchanger (NHE) activity was measured by monitoring changes in intracellular pH (pHi) after an acid load in a single cell level using the pH-sensitive dye 2'7'-bis-(2-carboxyethyl)-5.6'carboxyfluorescein (BCECF). Results: Amiloride and 5-(N-ethyl-N-isopropyl) amiloride (EIPA) inhibited the NHE activity with half-maximal inhibition values (IC50) of 15.3 and 4.0 muM, respectively. NHE-3 mRNA was detected by the RT-PCR technique in clonally proliferated PT cells. Conclusion: These results suggest that cultured PT cells isolated from human urine express amiloride-resistant NHE-3 activity on the apical membranes, which can be compared to functional properties of PT in vivo. Our experimental strategy offers a useful experimental approach to investigating human renal tubular transport function in vitro.