Regulation of opossum kidney (OK) cell Na/Pi cotransport by Pi deprivation involves mRNA stability.

Renal proximal tubular Na-dependent phosphate transport (Na/Pi cotransport) has been studied extensively in the opossum kidney (OK) cell line. Recently, we cloned a complementary deoxyribonucleic acid (cDNA) (NaPi-4) from OK cells encoding an apical NaPi cotransport system. OK cells exposed to a low-Pi medium, as compared to high-Pi media, responded with an increase in Na/Pi cotransport, which was followed by an increase in NaPi-4 messenger ribonucleic acid (mRNA) abundance; maximal stimulation of Na/Pi cotransport was reached in 2 h, with no further increase for up to 16 h. NAPi-4 mRNA abundance was unaltered for 2 h, then increased to a maximum after 6-16 h in cells treated with low Pi medium. NaPi-4 mRNA decay rate was lowered by low-Pi media when compared to high-Pi media, with no increase in the NaPi-4 mRNA transcription rate. These data suggest that the upregulation of Na/Pi cotransport in OK cells by low-Pi media involves two regulatory mechanisms: an immediate (early) increase (after 2 h) in the expression of Na/Pi cotransport, independent of mRNA synthesis or stability, and a delayed (late) effect (after 4-6 h), resulting in an increase in NaPi-4 mRNA abundance, due to an increased stability.

Cloning of a rabbit renal Na-Pi cotransporter, which is regulated by dietary phosphate.

Previously, we isolated a cDNA (NaPi-1) related to a rabbit renal proximal tubular Na-Pi cotransporter (A. Werner, M.L. Moore, N. Mantei, J. Biber, G. Semenza, and H. Murer. Proc. Natl. Acad. Sci. USA 88:9608-9612, 1991.). In this study, we isolated an additional (rabbit renal) cDNA (NaPi-6), which induces Na-dependent Pi uptake in Xenopus laevis oocytes. Substrate specificity and kinetic properties corresponded to those known for rabbit renal brush-border membrane (BBM) Na-Pi cotransport. NaPi-6 was cloned by homology using NaPi-2 cDNA, a rat renal BBM Na-Pi cotransporter (S. Magagnin, A. Werner, D. Markovich, V. Sorribas, G. Stange, J. Biber, and H. Murer. Proc. Natl. Acad. Sci. USA 90: 5979-5983, 1993). NaPi-6 encodes a protein of 642 amino acids, exhibiting at least eight transmembrane domains. NaPi-6 mRNA and protein in kidneys of rabbits fed a low-Pi diet (LPD; 0.11% Pi) for 1 wk were increased by 1.5- and 4-fold, respectively, compared with those of rabbits fed a high-Pi diet (HPD; 1.20% Pi). This effect was correlated with an increase in Na-Pi cotransport of BBM vesicles isolated from animals adapted to LPD (2.5-fold with respect to HPD). In contrast, NaPi-1 mRNA and protein were not altered in response to LPD. Thus rabbit proximal tubular BBMs contain two different Na-Pi cotransport systems: NaPi-1 (type I) and NaPi-6 (type II). Only the type II transport system seems to be under regulatory control in response to low-Pi dietary intake.

Expression of rat ileal Na(+)-sulphate cotransport in Xenopus laevis oocytes: functional characterization.

Small-intestinal sulphate absorption is a Na(+)-dependent process having its highest rate in the ileum; it involves brush-border membrane Na(+)-sulphate cotransport. Injection of rat ileal mRNA into Xenopus laevis oocytes induced Na(+)-dependent sulphate uptake in a dose-dependent manner, with no apparent effect on Na(+)-independent sulphate uptake. For mRNA-induced transport, the apparent Km value for sulphate interaction was 0.6 +/- 0.2 mM and that for sodium interaction was 25 +/- 2 mM (Hill coefficient: 2.3 +/- 0.3). mRNA-induced transport, was inhibited by thiosulphate, but not by phosphate or 4,4,'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). Using a rat renal Na(+)-sulphate cotransporter cDNA as a probe [NaSi-1; Markovich et al. (1993) Proc Natl Acad Sci USA 90:8073-8077], the highest hybridization signals (2.3 kb and 2.9 kb) were obtained in size fractions showing the highest expression of Na(+)-dependent sulphate transport in oocytes. Hybrid depletion experiments using antisense oligonucleotides (from the NaSi-1 cDNA sequence), provided further evidence that rat small-intestinal (ileal) Na(+)-sulphate cotransport is closely related to rat proximal-tubular brush-border membrane Na(+)-sulphate cotransport.