Renal Phosphate Transport Research Articles

Renal phosphate transport and vitamin D metabolism in X-linked hypophosphatemic Gy mice: responses to phosphate deprivation

Renal phosphate transport and vitamin D metabolism in X-linked hypophosphatemic Gy mice: responses to phosphate deprivation

Mechanism of dopamine inhibition of renal phosphate transport.

Dopamine (DA) is natriuretic and phosphaturic. However, whether the effect of DA on Pi reabsorption is a consequence of its effect on sodium transport is not known. Therefore, this study was performed to determine the effect of DA on the maximal transport of phosphate (TmPi), and upon the capacity of renal proximal brush border membrane (BBM) for (Naextra-vesicular greater than Naintravesicular)-gradient-dependent transport of Pi, as compared with the transport of other solutes. Graded infusions of Pi (0, 1, 2, and 3 mumols/min) were given to thyroparathyroidectomized male Sprague-Dawley rats in the presence of vehicle (0.9% NaCl; N = 5), DA 15 micrograms/kg/min; N = 6), or parathyroid hormone ((PTH); 1 U/kg/min; N = 5). The TmPi for rats infused with DA (3.3 +/- 0.3 mumol/mL) was significantly less than the TmPi for saline control rats (4.4 +/- 0.2 mumol/mL). Rats infused with PTH exhibited the lowest TmPi (1.8 +/- 0.3 mumol/mL). No differences in sodium excretion were observed among any of the groups. Na-dependent Pi transport was studied in BBM vesicles (BBMV) prepared from rats fed a low-phosphate diet for 2 days that were anesthetized, acutely thyroparathyroidectomized, and systemically infused with DA (350 micrograms bolus, plus 35 micrograms/kg/min; N = 8), PTH (33 U/kg bolus, followed by a continuous infusion of 1 U/kg/min; N = 6), or vehicle (1 mL/kg bolus, plus 2 mL/h constant infusion of 0.9% NaCl; N = 8) for 90 min. DA significantly inhibited the Na cotransport of Pi by 22.4 +/- 4.1% (P less than 0.01) as compared with the control group.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of essential arginine residues implicated in the renal transport of phosphate and glucose

We have characterized the reaction of arginine-specific reagents with the phosphate and glucose carriers of the kidney brush-border membrane. The inhibition of phosphate and glucose transport by phenylglyoxal follows pseudo-first-order kinetics. The rate of inactivation of phosphate transport by 50 mM phenylglyoxal was about 3-fold higher than that for glucose transport ( k app was 0.052 s −1 for the uptake of phosphate and 0.019 s −1 for the uptake of glucose). The order of the reaction, n, with respect to phenylglyoxal was 1.25 and 1.31 for the inactivation of phosphate and glucose transport, respectively. The inactivation of phosphate flux by p-hydroxyphenylglyoxal also follows pseudo-first-order kinetics, but the inhibition rate ( k app = 0.0012 s −1) was slower than with phenylglyoxal. The inactivation increased with the alkalinity of the preincubation medium for both phosphate and glucose fluxes and was maximal at pH 9.0. The inactivation of phosphate flux by phenylglyoxal depends upon the presence of an alkaline intravesicular pH. Extravesicular pH does not affect the reaction. Phenylglyoxal does not interfere with the recycling of the protonated carrier since phosphate uptake is inhibited independently of the pH used for transport measurements. Moreover, phenylglyoxal completely abolished trans stimulation by phosphate. Trans sodium inhibited phosphate uptake and abolished the pH profile of phosphate uptake.

Development of Tubular Function

This article has reviewed the maturation of major renal tubular transport systems. The tubular reabsorption of certain amino acids and the secretion of organic acids, hydrogen ions, and potassium increases as a function of postnatal age, being relatively immature at birth, especially in the preterm infant. In contrast, the ability for phosphate reabsorption is enhanced during the immature state as the developing animal attempts to adapt to its environment. In the case of glucose, the transport system is relatively mature in the term infant and less so in the infant of less than 34 weeks' gestation. One should consider these developmental changes in the renal tubular transport of amino acids, potassium, phosphate, and organic acids in the nutritional assessment and pharmacologic treatment of preterm as well as term infants.

Tyrosine phosphorylation selectively regulates renal cellular phosphate transport. Evidence that it mediates the stimulatory effect of insulin- like growth factor-1

The signal transduction mechanism responsible for the stimulation of the transport of inorganic phosphate (Pi) in response to mitogens is not known. In the present study, the changes in both Pi transport and tyrosine phosphorylation activity were determined in response to orthovanadate (VO4), an insulin-like agent and genistein, a specific inhibitor of tyrosine kinase activity. The results indicate that in opossum kidney epithelia, VO4 stimulated and genistein inhibited Pi transport dose dependently. The characteristics of the VO4 effect were quite similar to those described for insulin-like growth factor-1 (IGF-1) in terms of time course, selectivity (no effect on the Na-alanine transport), and dependency of the de novo synthesis of proteins. The effects of VO4 and IGF-1 on Pi transport, when tested at submaximal and maximal concentrations, respectively, were not additive suggesting that these two agents act through a common regulatory mechanism. As previously shown with IGF-1, the VO4 effect on Pi transport was additive to that of the maximal effect of Pi deprivation. Changes in tyrosine phosphorylation activity were tested in purified plasma membrane isolated from confluent OK cells using the polymer Glu:Tyr (4:1) as exogenous substrate. VO4 markedly enhanced whereas genistein inhibited the tyrosine kinase activity. The VO4 effect was dose dependent (0.1-1.0 mM), a concentration range similar to that eliciting the Pi transport response. The change in Pi transport was highly correlated with the variation in the tyrosine kinase activity induced by either vanadate (R = 0.969 P less than 0.01) or genistein (R = 0.992, P less than 0.01). In conclusion, VO4, an insulin-like agent and genistein, a specific inhibitor of tyrosine kinase activity selectively altered cellular Pi transport. These changes in Pi transport were associated with a dose-related alteration in tyrosine kinase activity measured in purified plasma membrane. The characteristics of the vanadate effects on Pi transport are similar to those reported for IGF-1 suggesting an important role of this signal transduction mechanism in mediating changes in Pi transport in response to mitogenic factors such as IGF-1.

Tyrosine phosphorylation selectively regulates renal cellular phosphate transport. Evidence that it mediates the stimulatory effect of insulin-like growth factor-1.

The signal transduction mechanism responsible for the stimulation of the transport of inorganic phosphate (Pi) in response to mitogens is not known. In the present study, the changes in both Pi transport and tyrosine phosphorylation activity were determined in response to orthovanadate (VO4), an insulin-like agent and genistein, a specific inhibitor of tyrosine kinase activity. The results indicate that in opossum kidney epithelia, VO4 stimulated and genistein inhibited Pi transport dose dependently. The characteristics of the VO4 effect were quite similar to those described for insulin-like growth factor-1 (IGF-1) in terms of time course, selectivity (no effect on the Na-alanine transport), and dependency of the de novo synthesis of proteins. The effects of VO4 and IGF-1 on Pi transport, when tested at submaximal and maximal concentrations, respectively, were not additive suggesting that these two agents act through a common regulatory mechanism. As previously shown with IGF-1, the VO4 effect on Pi transport was additive to that of the maximal effect of Pi deprivation. Changes in tyrosine phosphorylation activity were tested in purified plasma membrane isolated from confluent OK cells using the polymer Glu:Tyr (4:1) as exogenous substrate. VO4 markedly enhanced whereas genistein inhibited the tyrosine kinase activity. The VO4 effect was dose dependent (0.1-1.0 mM), a concentration range similar to that eliciting the Pi transport response. The change in Pi transport was highly correlated with the variation in the tyrosine kinase activity induced by either vanadate (R = 0.969 P less than 0.01) or genistein (R = 0.992, P less than 0.01). In conclusion, VO4, an insulin-like agent and genistein, a specific inhibitor of tyrosine kinase activity selectively altered cellular Pi transport. These changes in Pi transport were associated with a dose-related alteration in tyrosine kinase activity measured in purified plasma membrane. The characteristics of the vanadate effects on Pi transport are similar to those reported for IGF-1 suggesting an important role of this signal transduction mechanism in mediating changes in Pi transport in response to mitogenic factors such as IGF-1.

IGF-I, a Key Regulator of Renal Phosphate Transport and 1,25-Dihydroxyvitamin D3 Production During Growth

During development of vertebrates the extracellular concentration of Pi and, consequently, the amount of Pi available for cellular growth and bone mineral deposition is maintained at a higher level than during adult life. Insulin-like growth factor I plays a specific role in this Pi economy.

Effects of maleic acid on renal phosphorus transport: role of dietary phosphorus

The effects of maleic acid on renal phosphate (Pi) transport were examined by clearance and brush-border membrane vesicle (BBMV) transport studies. In normal rats, maleic acid 50 mg.kg body wt-1.h-1 increased the phosphaturia (P less than 0.001). Intraperitoneal administration of a similar dose of maleic acid decreased the BBMV uptake of Pi but not glucose. In rats fed a low-phosphate diet (0.03%), the maleic acid-induced phosphaturia was blunted, but the inhibitory effect on the BBMV transport of Pi persisted. In chronic parathyroidectomized rats fed a low-phosphate diet, where the filtered load of Pi was higher than in the previous groups, the phosphaturia was abolished, but the inhibition of the BBMV transport of Pi was sustained. Both the in vitro incubation of BBMVV and in vivo administration of maleic acid were associated with a competitive inhibition of Pi transport. These studies indicate that the maleic acid-induced phosphaturia is expressed at the apical membrane entry step of Pi, and the enhanced distal tubular reabsorption accounts for the lack of phosphaturia in dietary Pi deprivation.

Renal 25-hydroxyvitamin D-1 alpha-hydroxylase activity and mitochondrial phosphate transport in Hyp mice

The Hyp mouse is a homologue of the X chromosome-linked human disease, familial hypophosphatemic rickets (FHR). In FHR, reduced renal tubular brush-border membrane transport of phosphate results in hypophosphatemia and rickets. Both humans with FHR and Hyp mice have abnormal regulation of 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase), a mitochondrial enzyme found in proximal renal tubular cell epithelia, the apparent site of defective brush-border membrane phosphate transport. No common pathophysiology for these defects has been demonstrated. We hypothesized that phosphate transport may be present in renal mitochondria from Hyp mice and that its regulation may be deranged in parallel with the mitochondrial 1 alpha-hydroxylase. Using inhibitor-stop techniques described for measurement of phosphate transport in liver mitochondria, we examined mitochondria in normal and Hyp mouse kidney and found them to be comparable. We performed manipulations known to alter 1 alpha-hydroxylase differentially in normal and Hyp mice, i.e., phosphorus deprivation and phosphorus loading, and found no effect on mitochondrial phosphate transport. We also subjected Hyp and normal mice to calcium and vitamin D deprivation; this maneuver resulted in no significant changes in mitochondrial phosphate transport in Hyp or normal mice but confirmed the earlier observation that 1 alpha-hydroxylase activity is stimulated to a greater degree in normal mice than Hyp mice after this diet. Furthermore, administration of 1,25-hydroxyvitamin D3 depresses 1 alpha-hydroxylase activity in mitochondria from both normal and Hyp mice but has no effect on mitochondrial phosphate transport. We conclude that the mechanism of abnormal vitamin D metabolism in Hyp mice is not related to a primary defect in renal mitochondrial phosphate transport.

Conserved loci on the X chromosome confer phosphate homeostasis in mice and humans

Several genes expressed in kidney and other tissues determine phosphate homeostasis in extracellular fluid. The major form of inherited hypophosphatemia in humans involves an X-linked locus (HPDR, Xp22.31-p21.3). It has two murine homologues (Hyp and Gy) which map to closely-linked but separate loci (crossover value 0.4%-0.8%). Both murine mutations impair Na(+)-phosphate cotransport in renal brush border membrane; an associated renal disorder of 1,25-dihydroxyvitamin D3 (1,25(OH)2D) metabolism has been characterized in Hyp mice. Whereas experiments with cultured Hyp renal epithelium indicate that the gene is expressed in kidney, studies showing the development of the mutant renal phenotype in normal mice parabiosed to Hyp mice implicate a circulating factor; these findings can be reconciled if the humoral factor is of renal origin. The gene dose effect of HPDR, Hyp and Gy on serum phosphorus values is consistently deviant and heterozygotes resemble affected hemizygotes. The deviant effect is also seen on renal phosphate transport; all mutant females (Hyp/Hyp and Hyp/+) have similar phenotypes. On the other hand, there is a normal gene dose effect of HPDR in mineralized tissue; tooth PRATIO (pulp area/tooth area) values for heterozygotes are distributed between those for affected males and normals. The tooth data imply that the X chromosome locus is expressed in both renal and non-renal cells. The polypeptide product of the X chromosome gene(s) is still unknown.

Decreased concentration of 1,25-dihydroxyvitamin D 3 receptors in peripheral mononuclear cells of patients with X-linked hypophosphatemic rickets: effect of phosphate supplementation

Abnormal renal tubular phosphate transport is considered to be the primary defect in X-linked hypophosphatemic rickets (XLH). However, the resistance to vitamin D treatment in XLH cannot be explained by hypophosphatemia alone. Since most of the actions of vitamin D are mediated by its receptors (VDR), abnormalities of VDR have been postulated in XLH. In order to investigate this possibility, we measured the concentration of VDR in PHA-activated peripheral mononuclear cells from 10 XLH patients. Patients without phosphate supplementation showed significantly lower concentration (21.7 ± 5.1 fmol/mg protein, mean ± SEM) compared to the normal controls (60.7 ± 4.0). On the contrary, there was no significant difference between the phosphate-supplemented patients (58.3 ± 2.7) and controls. There was a significant positive correlation between VDR concentration and serum phosphate ( P < 0.05). In two patients, VDR was increased after daily phosphate supplementation was started. These results indicate that a decreased concentration of VDR secondary to persistent hypophosphatemia is one of the causes of vitamin D resistance in XLH.

Insulin stimulates phosphate transport in opossum kidney epithelial cells

Insulin is antiphosphaturic in vivo and this effect is due, in part, to increased Na(+)-dependent phosphate uptake across the luminal brush-border membrane of the proximal tubule. The intracellular mechanism is not understood. The present study shows that the stimulatory effect of insulin on phosphate transport can be reproduced in opossum kidney (OK) cells, suggesting that this established renal epithelial cell line may be a good model for further studies on insulin action on renal phosphate transport. The stimulation by insulin was dose related when insulin was used at concentrations within the range of 10(-14) to 10(-8) M. At 10(-8) M, insulin had no effect on Na(+)-independent uptake of phosphate or on the Na(+)-dependent uptakes of methyl-alpha-D-glucopyranoside and glutamate. The onset of insulin action on phosphate uptake was detected within 15 min, and the stimulation was reversed completely within 30 min after removal of insulin from the medium. Insulin action was not blocked by protein synthesis inhibitors and was not altered by bacitracin, an inhibitor of intracellular degradation of insulin. Pretreatment with the calcium-channel blockers, nifedipine and verapamil (10(-4) M), produced significant increases in the stimulatory effect of insulin, suggesting indirectly that insulin action on phosphate uptake may be influenced by Ca2+. In contrast to in vivo studies, there was no evidence that insulin interfered with parathyroid hormone action on OK cells.

Protein kinase C in mouse kidney: Effect of the Hyp mutation and phosphate deprivation

To test whether protein kinase C plays a role in the regulation of renal brush border membrane phosphate transport and mitochondrial vitamin D metabolism, we examined the activity, distribution and endogenous substrates of protein kinase C in renal subcellular fractions derived from two mouse models exhibiting perturbations in both renal functions. The X-linked Hyp mouse is characterized by reduced phosphate transport and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) synthesis relative to normal, whereas the phosphate-deprived mouse exhibits elevated phosphate transport and vitamin D hormone synthesis. Protein kinase C activity was higher in renal cytosol of Hyp mice, when compared to normal littermates (358 +/- 11 vs. 244 +/- 31 pmol 32P/mg prot/min, P less than 0.02), whereas genotype differences in brush border membrane and mitochondrial kinase were not apparent. Phosphate deprivation of normal mice elicited a 50% reduction in brush border membrane protein kinase C (from 819 +/- 56 to 460 +/- 48 pmol 32P/mg prot/min, P less than 0.03), an increase in mitochondrial kinase (from 57 +/- 7 to 87 +/- 10 pmol 32P/mg prot/min, P less than 0.03), and no change in cytosolic kinase activity. Phosphate deprivation of Hyp mice led to an increase in mitochondrial protein kinase C (from 72 +/- 7 to 98 +/- 9 pmol 32P/mg prot/min, P less than 0.03) and no change in either brush border membrane or cytosolic kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Southwestern Internal Medicine Conference: Clinical Disorders of Renal Tubular Phosphate Transport

Southwestern Internal Medicine Conference: Clinical Disorders of Renal Tubular Phosphate Transport

Phorbol myristate acetate activates protein kinase C, stimulates the phosphorylation of endogenous proteins and inhibits phosphate transport in mouse renal tubules

Calcium-activated, phospholipid-dependent protein kinase (protein kinase C) has been implicated in the regulation of transport processes in a variety of tissues and cell lines. To establish whether protein kinase C participates in the regulation of renal phosphate transport, we examined the effect of phorbol myristate acetate (PMA), a potent activator of protein kinase C, on phosphate uptake in fresh preparations of mouse renal tubules, and we correlated the changes in transport activity with protein kinase C activation and phosphorylation of endogenous proteins. PMA inhibited Na+-dependent phosphate transport, elicited a rapid translocation of protein kinase C from the cytosolic to the particulate fraction and stimulated the phosphorylation of endogenous substrates in the cytosolic and brush border membrane fractions. Effects of PMA were maximal after a 10 min incubation of the tubules with the activator. 4 alpha-Phorbol, an inert analogue of PMA, did not elicit any of these effects. The present results demonstrate a temporal correlation between inhibition of Na+-dependent phosphate transport, translocation and activation of protein kinase C, and phosphorylation of endogenous proteins in mouse renal tubules. These data suggest that protein kinase C may play a regulatory role in phosphate transport in mammalian kidney.

The renal phosphate transport defect in normal mice parabiosed to X-linked hypophosphatemic mice persists after parathyroidectomy.

The X-linked hypophosphatemic (Hyp) mouse is a model for human X-linked hypophosphatemia. Surgical joining of normal to Hyp mice by parabiosis results in the normal mice developing low renal retention of phosphate and hypophosphatemia. These results suggest a humoral component to the renal defect. To test whether this component could be parathyroid hormone, surgical parathyroidectomy (PTX) or sham surgery was performed in mice 3 weeks after parabiotic union (n greater than 20 per group). After an overnight fast, PTX mice were hypocalcemic and hyperphosphatemic relative to sham-operated control mice. PTX normal mice joined to PTX Hyp mice were significantly lower in plasma phosphate and higher in fractional excretion of phosphate [U/P phosphate/(U/P creatinine)] when compared with PTX normal mice joined to other PTX normals. To test for more specific evidence of altered renal transport function, renal brush-border membrane vesicles (BBMV) were prepared from these mice, and phosphate and glucose uptakes were measured. The phosphate/glucose transport ratio was lower in BBMV from Hyp mice, joined to either normal mice or to Hyp mice, when compared with that from normal-normal pairs. Moreover, BBMV from normal mice joined to Hyp mice had a significantly lower phosphate/glucose uptake ratio than BBMV from normal mice joined to other normal mice, and their activity approached that of BBMV derived from Hyp mice. Glucose uptake in BBMV was unaffected by parabiosis or genotype. In summary, parabiosis of normal mice to Hyp mice resulted in the development of phosphaturia and decreased BBMV phosphate transport in the normal mice. The persistence of the phosphate transport defect in parathyroidectomized mice suggests that parathyroid hormone is not the humoral factor contributing to these results.

Role of BBM lipid composition and fluidity in impaired renal Pi transport in aged rat.

Renal tubular phosphate (Pi) transport is impaired in the aged rat. The decrement in Pi transport is manifested as a decrease in the Vmax of the luminal brush-border membrane (BBM) sodium-dependent Pi transport. In the present study we determined the potential role of alterations in cortical BBM lipid composition and fluidity in the age-related phosphaturia observed in rats. In the aged rat there are significant increases in the BBM cholesterol (Chol, 504 vs. 422 nmol/mg protein in adult, P less than 0.01) and sphingomyelin (Sph, 41.8 vs. 37.5 mol% in adult, P less than 0.01) and a decrease in the BBM fluidity [increase in fluorescence anisotropy of diphenylhexatriene (DPH), rDPH, 0.221 vs. 0.215 in adult, P less than 0.01]. The BBM lipid compositional and fluidity alterations may also play an important role in the impaired renal adaptation to a low-Pi diet in the aged rat. In the adult rat the renal adaptation to a low-Pi diet is associated with a decrease in BBM Chol (370 to 307 nmol/mg protein, P less than 0.01) and an increase in fluidity (decrease in rDPH 0.207 to 0.201, P less than 0.05). In the aged rat the renal adaptation to a low-Pi diet is incomplete and is associated with impaired ability to lower BBM Chol (441 to 429 nmol/mg protein, P = NS) and to increase fluidity (rDPH 0.211 to 0.211, P = NS). The results of this study therefore suggest that in the aged rat the increase in BBM Chol and Sph content, and the decrease in BBM fluidity play an important role in the impaired renal tubular Pi transport and adaptation to a low-Pi diet.

Renal phosphate transport in humoral hypercalcemia of malignancy.

Fisher rats bearing the H-500 Leydig cell tumor (LCT) develop humoral hypercalcemia of malignancy (HHM), which is accompanied by hypophosphatemia and hyperphosphaturia. To better define the mechanisms underlying the changes in phosphate metabolism, the activity of sodium-dependent phosphate uptake (Na+-Pi) in microvillus membrane vesicles (MMV) isolated from the renal cortex of LCT-bearing rats was studied. Ten days after tumor transplantation the animals became hypercalcemic, hypophosphatemic, and hyperphosphaturic, and LCT-MMV showed a specific decrease in Na+-Pi. A kinetic analysis revealed evidence for both a high- and a low-affinity system of Na+-Pi. The Vmax of both the low-affinity system and the high-affinity system were significantly reduced in LCT-MMV. These changes in Na+-Pi transport were similar to those induced by parathyroid hormone. In day-10 tumor-bearing animals, daily injections of dichloromethylene diphosphonate (2.5 mg.kg-1.day-1) prevented the onset of hypercalcemia but not the reduction in Na+-Pi in LCT-MMV. Our data suggest that in this animal model of HHM there is a specific and persistent impairment of Na+-Pi uptake at the level of the renal cortical brush-border membrane, which contributes to the derangement in phosphate metabolism.

Correlations of Serum Concentrations of 1,25-Dihydroxyvitamin D, Phosphorus, and Parathyroid Hormone in Tumoral Calcinosis*

The inherited metabolic disorder tumoral calcinosis is characterized by elevated serum phosphorus and 1,25-dihydroxyvitamin D [1,25-(OH)2D] levels and paraarticular calcific tumors. The pathogenesis of this disease is obscure, but an elevated renal phosphate reabsorption threshold and increased production of 1,25-(OH)2D are postulated as defects. We studied nine affected patients and found that both serum phosphorus and renal phosphate reabsorption threshold (TmP/GFR) were positively correlated with serum 1,25-(OH)2D levels. Since tumoral calcinosis is a disorder with abnormal renal phosphate transport, we compared the TmP/GFR and serum 1,25-(OH)2D levels to values obtained in patients with two other diseases with renal phosphate transport defects: oncogenic osteomalacia and X-linked hypophosphatemic rickets. We found a significant correlation between TmP/GFR and 1,25-(OH)2D levels in all three diseases, suggesting that in these diseases 1,25-(OH)2D production is regulated in some manner by phosphate transport. Furthermore, previous work indicated that in tumoral calcinosis broad variation exists in serum phosphorus levels. In our patients a negative correlation was found between the serum PTH concentrations and both serum phosphorus levels and TmP/GFR values, respectively. We postulate that although the basic defect in tumoral calcinosis most likely resides in the proximal renal tubular cell, the variation in serum phosphorus levels and possibly disease expression is modulated in part by PTH.

Nicotinamide as a rapid-acting inhibitor of renal brush-border phosphate transport

Administration of nicotinamide to rats produces specific dose-dependent inhibition of Na+-dependent phosphate transport across the renal brush-border membrane (BBM) and an increase in urinary excretion of phosphate. The intracellular mechanism of action of nicotinamide is not well established. As a step in this direction, the present studies determined whether nicotinamide was a rapid- or slow-acting regulator of the BBM phosphate transport system. Nicotinamide (0.5 g/kg) inhibited Na+-dependent BBM phosphate transport under conditions when de novo protein synthesis was inhibited by cycloheximide (1.0 mg/kg). Furthermore, the degree of inhibition was not different from that achieved by nicotinamide alone, suggesting that the action of nicotinamide does not require de novo protein synthesis. Studies on the time course of the onset of nicotinamide action revealed inhibition of BBM phosphate transport within 1 h after injection of nicotinamide, even in rats pretreated with cycloheximide. The rapid response to nicotinamide and its independence of de novo protein synthesis characterize nicotinamide as a rapid-acting regulator of the Na+-dependent phosphate transport system in renal BBM.