Effect of high osmolality on Na+/H+ exchange in renal proximal tubule cells.
Na+/H+ exchanger isoform and the effect of high osmolality on its function was studied in cultured renal epithelial cells (LLC-PK1 and OK). Using NHE-3-specific antibody, immunoblots of luminal membranes from LLC-PK1 and OK cells specifically labeled proteins with molecular masses 90 and 95 kDa, indicating that NHE-3 is the isoform expressed on the luminal membranes of these epithelia. Proximal tubular suspensions from rabbit kidney cortex were incubated in control (310 mosm/liter) or high osmolality (510 mosm/liter) medium for 45 min and utilized for brush border membrane vesicle preparation. Influx of amiloride-sensitive 22Na+ at 10 s (pHo 7.5, pHi 6.0) into brush border membrane vesicles was 37% lower in the high osmolality group (p < 0.03). LLC-PK1 or OK cells were grown to confluence and examined for Na+/H+ exchange activity. An increase in medium osmolality to 510 mosm following acid loading decreased the 5-min uptake of the amiloride-sensitive 22Na+ in LLC-PK1 and OK cells (p < 0.04 and < 0.03 for LLC-PK1 cell OK cells, respectively). An increase in medium osmolality to 510 mosm in vascular smooth muscle cells, which express NHE-1, produced 45 and 64% stimulation of the amiloride-sensitive 22Na+ influx at base-line pHi and acid-loaded condition, respectively (p < 0.03 and < 0.01). Down-regulation of protein kinase C by preincubation with phorbol 12-myristate 13-acetate or inhibition of Ca(2+)-calmodulin-dependent protein kinase (calmodulin-kinase II) by N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide (W-7) in LLC-PK1 cells did not block the inhibitory effect of high osmolality on Na+/H+ exchange activity. We conclude that renal proximal tubule epithelial cells express Na+/H+ exchange isoform NHE-3 on their luminal membranes and that hyperosmolality decreases transporter activity during cell acidification. This inhibitory effect might be unique to the NHE-3 isoform, since vascular smooth muscle cells which express NHE-1 exhibit an increase in Na+/H+ exchange activity in response to high osmolality.
Soleimani
M
M
Department of Medicine, Indiana University, Indianapolis 46202-5116.
Bookstein
C
C
McAteer
J A
JA
Hattabaugh
Y J
YJ
Bizal
G L
GL
Musch
M W
MW
Villereal
M
M
Rao
M C
MC
Howard
R L
RL
Chang
E B
EB
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006982:Hypertonic Solutions; D015151:Immunoblotting; D007672:Kidney Cortex / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*
D006982:Hypertonic Solutions; D011993:Recombinant Fusion Proteins; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C039465:5-dimethylamiloride; D000584:Amiloride; D012964:Sodium