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 microM, 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.
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 microM, 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.
Authors: John J Gildea; Dylan T Lahiff; Robert E Van Sciver; Ryan S Weiss; Neema Shah; Helen E McGrath; Cynthia D Schoeffel; Pedro A Jose; Robert M Carey; Robin A Felder Journal: Clin Chim Acta Date: 2013-02-27 Impact factor: 3.786