Abstract
Chronic dietary phosphate restriction is associated with up-regulation of sodium-dependent phosphate (Na/Pi) cotransport by renal proximal tubular epithelial cells in association with increases in Na/Pi cotransporter mRNA and protein. We investigated whether changes in cytosolic calcium mediate this adaptive response in opossum kidney cells, a continuous line of renal epithelial cells. After 24 h of phosphate depletion, steady-state cytosolic calcium levels were increased; this increase was observed at physiologic levels of phosphate restriction and was prevented by the calcium channel blocker verapamil. Chronic phosphate depletion was also associated with parallel increases in Na/Pi cotransport activity, Na/Pi cotransporter mRNA, and Na/Pi cotransporter protein, all of which were blocked in verapamil-treated cells. Actinomycin D, at a dose that prevented the increase in NaPi-4 mRNA during phosphate depletion, also prevented the increase in Na/Pi cotransport activity. Incubation with the calcium ionophore ionomycin or A23187 reproduced the increase in Na/Pi cotransporter mRNA in phosphate-replete cells. Conversely, chelation of cytosolic calcium by quin-2/AM prevented the increase in Na/Pi cotransporter mRNA in phosphate-depleted cells. The effect of an increase in cytosolic calcium was specific for the Na/Pi cotransporter as mRNA levels for the sodium-dependent glucose transporter were not affected. Our observations suggest that chronic phosphate restriction increases steady-state cytosolic calcium, which, in turn, increases transcription of Na/Pi cotransporter mRNA, thereby stimulating Na/Pi cotransport activity.
Highlights
Chronic dietary phosphate restriction is associated with up-regulation of sodium-dependent phosphate (Na/ Pi) cotransport by renal proximal tubular epithelial cells in association with increases in Na/Pi cotransporter mRNA and protein
We have recently demonstrated in Opossum kidney (OK) cells parallel increases in Na/Pi cotransport activity, Na/Pi cotransporter NaPi-4 mRNA, and NaPi-4 protein during chronic Pi restriction (12), similar to the changes observed in Pi-deprived rats (6, 7)
Changes in Cytosolic Calcium during Phosphate Depletion— Incubation of OK cells in a Pi-free medium for 24 h was associated with an approximate doubling of steady-state cytosolic calcium levels, as compared with Pi-replete cells (Fig. 1)
Summary
Materials—OK cells were a gift of Judith Cole (University of Missouri, Columbia, MO). Culture media and dishes were from Life Technologies, Inc. Fura-2/AM was from Teflabs (Austin, TX). The verapamil enantiomers S(Ϫ)-verapamil and R(ϩ)-verapamil were from Research Biochemicals Inc. Enhanced chemiluminescent (ECL) kits for developing the Western blots were from Amersham (Buckinghamshire, United Kingdom).
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