Hypophosphatemia leads to an increase in type II Na؉ -dependent inorganic phosphate cotransporter (NaPi-2) mRNA and protein levels in the kidney and increases renal phosphate reabsorption. Nuclear transcript run-on experiments showed that the effect of a low phosphate diet was post-transcriptional. In an in vitro degradation assay, renal proteins from hypophosphatemic rats stabilized the NaPi-2 transcript 6-fold compared with control rats and this was dependent upon an intact NaPi-2 3-untranslated region (UTR). To determine an effect of hypophosphatemia upon NaPi-2 protein synthesis, the incorporation of injected [35 S]methionine into renal proteins was studied in vivo. Hypophosphatemia led to increased [ 35 S]methionine incorporation only into NaPi-2 protein. The effect of hypophosphatemia on translation was studied in an in vitro translation assay, where hypophosphatemic renal proteins led to increased translation of NaPi-2 and other transcripts. NaPi-2 RNA interaction with cytosolic proteins was studied by UV cross-linking and Northwestern gels. Hypophosphatemic proteins led to increased binding of renal cytosolic proteins to the 5-UTR of NaPi-2 mRNA. Therefore, hypophosphatemia increases NaPi-2 gene expression post-transcriptionally, which correlates with a more stable transcript mediated by the 3-UTR, and an increase in NaPi-2 translation involving protein binding to the 5-UTR. These findings show that phosphate regulates gene expression by affecting protein-RNA interactions in vivo.Dietary phosphorus is converted in the body to the phosphates, in which form it exerts its widespread physiological functions, as an essential component of phospholipids, ATP, DNA, phosphorylated proteins, metabolic intermediaries, body buffers, and bone (1). The renal tubule has an intrinsic ability to adjust the reabsorption rate of phosphate according to the need and availability of phosphate to the body. The renal tubule responds to a decrease in filtered phosphate load with an increase in transport activity, thereby maintaining renal phosphate homeostasis. The active reabsorption is mediated by the Na ϩ -dependent inorganic phosphate cotransporters (NaPi).1 NaPi type II are expressed at the apical brush border membranes of the proximal tubules and are predominantly responsible for the regulated reabsorption of phosphate in response to changes in dietary phosphate (2-4). In the rat, NaPi type II is termed NaPi-2. Deletion of the NaPi-2 gene in mice leads to severe phosphate wasting (5). The activity of NaPi-2 is increased by a low phosphate diet (6 -8) and after parathyroidectomy (9) (10) and decreased by a high phosphate diet, parathyroid hormone (9), glucocorticoids (11, 12), and metabolic acidosis (13,14). The increase in NaPi-2 is the result of an increase in V max , suggesting an increase in the number of apical NaPi-2 transporters, by a transporter shuttling mechanism, which is sensitive to disrupters of microtubule integrity (13, 15). A low phosphate diet for as little as 2 h led to an increased transfer of preformed ...
Abstract. The sensing and response to extracellular phosphate (Pi) concentration is preserved from prokaryotes to mammals and ensures an adequate supply of Pi in the face of large differences in its availability. In mammals, the kidneys are central to Pi homeostasis. Renal Pi reabsorption is mediated by a Na/Pi co-transporter that is regulated by a renal Pi sensing system and humoral factors. The signal transduction by which Pi regulates type II Na/Pi activity is largely unknown. It is shown that calcineurin inhibitors specifically and dramatically decrease type II Na/Pi gene expression in a proximal tubule cell line and in vivo. Mice with genetic deletion of the calcineurin A gene had a marked decrease in type II Na/Pi mRNA levels and remarkably did not show the expected increase in type II Na/Pi mRNA levels after the challenge of a low-Pi diet. In contrast, the regulation of renal 25(OH)-vitamin D 1␣-hydroxylase gene expression by Pi was intact. This is the first demonstration that calcineurin has a crucial role in the signal transduction pathway regulating renal Pi homeostasis both in vitro and in vivo. These results suggest that the use of calcineurin inhibitors contributes to the renal Pi wasting seen in renal transplant patients.Phosphate (Pi) homeostasis is essential to life and is dependent on active renal reabsorption. In diseases of phosphorus (P) homeostasis, such as X-linked hypophosphatemia or oncogenic osteomalacia, there is a tremendous renal P loss with severe bone disease (1). In contrast, in chronic renal failure, the P retention leads to secondary hyperparathyroidism with disabling bone disease and vascular calcification associated with a high mortality (2). The kidney has an intrinsic P sensing system that regulates the activity of apical brush border membrane type II Na/Pi co-transporters (3). However, it is still not known how the organism senses changes in serum P.There are three mammalian Na/Pi co-transporter families: types I through III. Type II Na/Pi activity is responsible for Ͼ80% of renal P reabsorption and contains three isoforms-IIa through c-of which IIa is the major factor in renal P reabsorption and regulation in adult mice (4). Type II Na/Pi activity is increased by a low serum P and decreased by parathyroid hormone (PTH) and FGF23 (4 -8). The regulation of type II Na/Pi is at the level of recruitment of new transporters to the apical proximal tubule membrane, the level of synthesis of new transporters and their breakdown (5,9). In addition, there is regulation at the level of type II Na/Pi gene expression, which in vivo is mainly posttranscriptional (10,11). In the rat, a cis acting element in the type II Na/Pi co-transporter (Na/Pi-2) mRNA at the junction of the coding region and the 3'-untranslated region (UTR) interacts with trans acting renal cytosolic proteins and determines Na/Pi-2 mRNA stability in response to dietary P restriction (12). An additional level of regulation of Na/Pi-2 is its translational control by a low-P diet (10).The renal type II Na/Pi co-transporte...
Moz, Yulia, Justin Silver, and Tally Naveh-Many. Characterization of cis-acting element in renal NaPi-2 cotransporter mRNA that determines mRNA stability.
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