These observations suggest that nicotinamide may regulate the expression of a major functional Na/Pi cotransporter in the rat small intestine.

Alteration of the dietary intake of phosphate (P(i)) leads to rapid changes in renal P(i) transport activity. The present study, examined the underlying cellular mechanisms of the rapid regulation, with special reference to renal P(i) cotransporter. Rats were fed either a low-P(i) (0.02%) diet (CLP rats), the low-P(i) diet followed by a high-P(i) (1.2%) diet (AHP rats), or a normal (0.6%) diet (control rats). Na(+)-dependent P(i) transport activity in the brush border membrane was significantly increased in CLP rats compared with control rats, and this activity decreased rapidly within 2 h after the change of diet in AHP rats. Kinetic analysis of P(i) transport in the AHP rats indicated that the reduction was accompanied by a decrease in the apparent Vmax for Na(+)-dependent P(i) uptake. Northern blot analysis showed no difference in the abundance of NaP(i)-2 mRNA of the kidney between AHP and CLP rats. In contrast, Western blot analysis of renal brush border membrane proteins of AHP rats indicated a significant decrease in the abundance of NaP(i)-2 protein as compared with CLP rats. Immunoreactive signals for NaP(i)-2 were detected in lysosomal fractions of AHP and CLP rats. Immunohistochemical analysis showed that, NaP(i)-2 immunoreactivity in AHP rats was largely reduced in the apical membrane of the proximal tubular epithelial cells. Neither cycloheximide nor actinomycin D affected high-P(i)-induced reduction of NaP(i)-2 protein in the brush border membrane of AHP rats, indicating that de novo protein synthesis of an unidentified regulator protein was not involved in the mechanism of this reduction. In contrast, treatment with colchicine, which disrupts microtubulers, abolished the effect of high-P(i) diet on NaP(i)-2 expression. These results suggested that rapid endocytotic internalization of NaP(i)-2 may occur specifically in the brush border membrane following an acute increase in dietary P(i) intake.

In a study of the rat intestinal P(i) transport system, an activator protein for rat Na/P(i) co-transport system (PiUS) was isolated and characterized. We also investigated the effects of restriction of vitamin D and P(i) (two of the most important physiological and pathophysiological regulators of P(i) absorption in the small intestine) on intestinal P(i) transport activity and the expression of Na/P(i) co-transporters that are expressed in rat small intestine. Rat PiUS encodes a 424-residue protein with a calculated molecular mass of 51463 Da. The microinjection of rat PiUS into Xenopus oocytes markedly stimulated Na(+)-dependent P(i) co-transport activity. In rats fed with a low-P(i) diet, Na(+)-dependent P(i) co-transport activity was increased approx. 2-fold compared with that of rats fed a normal P(i) diet. Kinetic studies demonstrated that this increased activity was due to an elevation of V(max) but not K(m). The PiUS mRNA levels showed an approximate doubling in the rats fed with the low-P(i) diet compared with those fed with the normal P(i) diet. In addition, after the administration of 1, 25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] to vitamin D-deficient animals, the P(i) uptake was significantly increased in the Na(+)-dependent component in the brush border membrane vesicle (BBMV) at 24 and 48 h. In addition, we found a further high-affinity Na/P(i) co-transport system in the BBMV isolated from the vitamin D-replete animals. The levels of type III Na/P(i) co-transporter PiT-2 mRNA were increased 24 and 48 h after 1,25-(OH)(2)D(3) administration to vitamin D-deficient animals, whereas PiUS and the type IIb Na/P(i) co-transporter mRNA levels were unchanged. In conclusion, we first cloned a rat activator protein, PiUS, and then studied its role along with that of other type III Na/P(i) co-transporters. PiUS and PiT-2 might be important components in the regulation of the intestinal P(i) transport system by P(i) restriction and 1,25-(OH)(2)D(3).

No abstract

Three lambda phage clones encompassing the Na+/phosphate co-transporter (NaPi-3) gene and its 5' flanking region were isolated from a human genomic DNA library. The gene comprises 13 exons and 12 introns and spans approx. 14 kb. All exon-intron junctions conform to the GT/AG rule. The major transcription-initiation site was determined by primer-extension analysis and is an adenosine residue 57 bp upstream of the 3' end of the first exon. There is a typical TATA box 28 bp upstream of the major transcription-initiation site and various cis-acting elements, including a cAMP-responsive element, AP-1, AP-2 and SP-1 sites in the 5' flanking region. This region also contains three direct-repeat-like sequences that resemble the consensus binding sequence for members of the steroid-thyroid hormone receptor superfamily, including vitamin D. Deletion analysis suggests that the region from nt-2409 to nt-1259 in the 5' flanking region may be involved in kidney-specific gene expression. Vitamin D responsiveness of the NaPi-3 promoter was also detected in COS-7 cells co-transfected with a human vitamin D receptor expression vector. The presence of the three vitamin D receptor- responsive elements in the NaPi-3 promoter may be important in mediating the enhanced expression of the gene by 1,25-dihydroxyvitamin D3.