Growth is critically dependent on the retention of a variety of nutrients. The kidney contributes to this positive external balance. In the present study, we isolated a cDNA from the human and rat kidney that encodes a growth-related Na ؉ -dependent inorganic phosphate (P i ) cotransporter (type IIc). Microinjection of type IIc cRNA into Xenopus oocytes demonstrated sodium-dependent P i cotransport activity. Affinity for P i was 0.07 mM in 100 mM Na ؉ . The transport activity was dependent on extracellular pH. In electrophysiological studies, type IIc Na/P i cotransport was electroneutral, whereas type IIa was highly electrogenic. In Northern blotting analysis, the type IIc transcript was only expressed in the kidney and highly in weaning animals. In immunohistochemical analysis, the type IIc protein was shown to be localized at the apical membrane of the proximal tubular cells in superficial and midcortical nephrons of weaning rat kidney. Hybrid depletion experiments suggested that type IIc could function as a Na/P i cotransporter in weaning animals, but its role is reduced in adults. The finding of the present study suggest that the type IIc is a growth-related renal Na/P i cotransporter, which has a high affinity for P i and is electroneutral.

We have demonstrated previously that the type-IIc Na/Pi cotransporter is a growth-related renal Na/Pi cotransporter that is highly expressed in kidney of the weaning rat. In the present study, we investigated type-IIc Na/Pi cotransporter function further by cloning the mouse gene and characterizing the corresponding protein. The mouse type-IIc transporter amino acid sequence shows a high degree of similarity to the human (86%) and rat (95%) type-IIc Na/Pi-cotransporters. The mouse gene contained 14 exons and mapped to chromosome 2. The DNA sequence upstream from exon 1 is GC rich. The upstream region does not contain an apparent TATA box, but does contain two dietary Pi-responsive elements, which are potential binding sites for the transcription factor micro E3 (TFE3). Microinjection of mouse type-IIc cRNA into Xenopus oocytes demonstrated sodium-dependent Pi cotransport activity. The affinity for Pi was about 200 microM in 100 mM Na. Feeding adult mice fed a low-Pi diet increased the expression of type-IIc protein in the apical membrane of renal proximal tubular cells. Hybrid depletion studies suggested that the type-IIc transporter contributes to about 30% of Na/Pi cotransport in the kidney of adult mice fed a low-Pi diet. The present study suggests that the type-IIc Na/Pi cotransporter is a functional of renal Pi transporter in adult mice fed a low-Pi diet.

The TGF-β superfamily comprises pleiotropic cytokines that regulate SMAD and non-SMAD signaling. TGF-β-SMAD signal transduction is known to be involved in tissue fibrosis, including renal fibrosis. Here, we found that 1,25-dihydroxyvitamin D 3 -bound [1,25(OH) 2 D 3 -bound] vitamin D receptor (VDR) specifically inhibits TGF-β-SMAD signal transduction through direct interaction with SMAD3. In mouse models of tissue fibrosis, 1,25(OH) 2 D 3 treatment prevented renal fibrosis through the suppression of TGF-β-SMAD signal transduction. Based on the structure of the VDR-ligand complex, we generated 2 synthetic ligands. These ligands selectively inhibited TGF-β-SMAD signal transduction without activating VDR-mediated transcription and significantly attenuated renal fibrosis in mice. These results indicate that 1,25(OH) 2 D 3 -dependent suppression of TGF-β-SMAD signal transduction is independent of VDR-mediated transcriptional activity. In addition, these ligands did not cause hypercalcemia resulting from stimulation of the transcriptional activity of the VDR. Thus, our study provides a new strategy for generating chemical compounds that specifically inhibit TGF-β-SMAD signal transduction. Since TGF-β-SMAD signal transduction is reportedly involved in several disorders, our results will aid in the development of new drugs that do not cause detectable adverse effects, such as hypercalcemia.

Dietary phosphate (P i ) is a most important regulator for renal P i reabsorption. The type II sodium-dependent phosphate (Na/P i ) cotransporters (NPT2) are located at the apical membranes of renal proximal tubular cells and major functional transporters associated with renal P i reabsorption. The consumption of a low-P i diet induces the synthesis of NPT2, whereas a high P i diet decreases it. The molecular mechanisms of regulation by dietary P i are not yet known.In this report, in weaning mice fed a low-P i diet for 4 days, the NPT2 mRNA level was increased 1.8-fold compared with mice fed a normal P i diet. This increase was due to an elevation of the transcriptional activity. In the NPT2 gene promoter, the DNA footprint analysis showed that six regions were masked by the binding protein, but at the position ؊1010 to ؊985 upstream of the transcription start site, the binding clearly responded to the levels of dietary P i . The phosphate response element (PRE) of the NPT2 gene was found to consist of the motif related to the E box, 5-CACGTG-3. A yeast one-hybrid system was used to clone a transcription factor that binds to the PRE sequences in the proximal promoter of the NPT2 gene. Two cDNA clones that encoded protein of the mouse transcription factor E3 (TFE3) were isolated. This is a DNA-binding protein that activates transcription through the E3 site of the immunoglobulin heavy chain enhancer. TFE3 antibody completely inhibited the binding to the PRE. The coexpression of TFE3 in COS-7 cells transfected with the NPT2 gene promoter markedly stimulated the transcriptional activity. The feeding of a low P i diet significantly increased the amount of TFE3 mRNA in the kidney. These findings suggest that TFE3 may participate in the transcriptional regulation of the NPT2 gene by dietary P i .The regulation of inorganic phosphate (P i ) 1 in the human body is controlled mainly by reabsorption in the proximal tubules of the kidneys (1-3). Apical Na ϩ -dependent phosphate (NaP i ) cotransport is central to the renal proximal tubular reabsorption of P i (2, 3). Studies of isolated kidney tubules and of brush-border membranes have demonstrated that the physiological regulation of proximal tubule P i transport involves complex hormonal and metabolic factors that affect the activity or expression of the transporter molecules (1-3). A major regulator of the NaP i cotransporter is dietary P i (4 -7). Dietary P i restriction is associated with an adaptive increase of the overall proximal tubular capacity to reabsorb P i (4 -7). Alterations of the dietary intake of P i lead to an adaptation of renal P i transport activity independent of extrarenal factors such as parathyroid hormone, growth hormone, and vitamin D (1-3, 9, 10). The molecular mechanisms of this adaptation are unknown.Three types of Na ϩ -dependent P i cotransporter have been isolated from a kidney library (11,12). Recent studies suggest that the type II transporters (NPT2) may play an important role in P i homeostasis in the kidney, that they are controlled b...