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


CONCLUSIONS
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.
Inoue
C N
CN
Department of Pediatrics, Tohoku University School of Medicine, Sendai, Japan.
Kondo
Y
Y
Ohnuma
S
S
Morimoto
T
T
Nishio
T
T
Iinuma
K
K
D000328:Adult; D001483:Base Sequence; D001692:Biological Transport; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D002675:Child, Preschool; D017931:DNA Primers; D006651:Histocytochemistry; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000378:metabolism; D008297:Male; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D014556:Urine / Q000166:cytology; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D017931:DNA Primers; D017923:Sodium-Hydrogen Exchangers; D005723:gamma-Glutamyltransferase