Dependent Phosphate Transport Research Articles

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.

Initiation and characterization of primary mouse kidney epithelial cultures

Primary cultures of murine renal epithelial cells were established from a preparation of proximal tubule fragments. Confluent cultures exhibited multiple dome formation, indicating the presence of tight junctions and an intact transcellular transport process. Ultrastructural analysis revealed a monolayer of polarized cells, with a sparse but clearly defined microvillar surface facing the growth medium and a basolateral surface attached to the substratum. Cultures grown on collagen gels did not show domes. The epithelial monolayer exhibited several differentiated functions of the proximal tubule: a) parathyroid hormone (PTH)-stimulated cAMP synthesis; b) production of 24,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3; c) high alkaline phosphatase activity; and d) Na+-dependent transport of phosphate (Pi) and alpha-methylglucoside (alpha-MG). The sugar uptake was selectively inhibited by phlorizin, a competitive inhibitor of glucose uptake at the luminal membrane. Kinetic analysis revealed independent transport systems for Pi and alpha-MG, with Km values corresponding to the high affinity systems identified in brush border membrane vesicles derived from the proximal tubule. Pi uptake by the epithelial monolayers was regulated by the concentration of Pi in the growth medium. Phorbol esters and PTH did not exert an effect on Pi and alpha-MG transport in mouse primary cultures. The present study demonstrates that primary cultures provide a useful in vitro preparation to investigate renal proximal tubular function.

Regulation of Sodium-Dependent Phosphate Transport by Parathyroid Hormone in Opossum Kidney Cells: Adenosine 3 ′,5 ′-Monophosphate-Dependent and -Independent Mechanisms*

The hormonal regulation of Na+-dependent phosphate transport was studied in opossum kidney (OK) cells. PTH caused time- and concentration-dependent decreases in Na+-dependent phosphate transport, with 10 pM PTH-(1-34) producing a 19% decline in phosphate transport. The EC50 for PTH inhibition of phosphate transport was 50 pM. Kinetic analyses of phosphate transport indicated that PTH decreased the maximum velocity without affecting the Km for phosphate. PTH increased cAMP formation with an EC50 of 10 nM. 8-Bromo-cAMP and (Bu)2cAMP also inhibited phosphate transport. Forskolin increased cAMP formation and decreased phosphate transport, whereas the cyclase-inactive forskolin analog 1,9-dideoxyforskolin also inhibited phosphate transport. The PTH analog [8,18-norleucine,34-tyrosinamide]PTH-(3-34) reduced phosphate transport at concentrations from 10 nM to 30 microM, but did not increase cAMP formation at concentrations up to 10 microM. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine produced concentration-dependent decreases in PTH-stimulated cAMP formation, but did not influence PTH inhibition of Na+-dependent phosphate transport. Vasoactive intestinal polypeptide and prostaglandin E1 increased cAMP formation in OK cells, but were weak inhibitors of phosphate transport. This study suggests that cAMP may not be the only transmembrane signaling mechanism involved in the regulation of Na+-dependent phosphate transport by PTH-(1-34) in OK cells.

Vitamin D metabolism and phosphate transport in developing kidney: effect of diet and mutation

In order to obtain a better understanding of the molecular mechanisms involved in phosphate reabsorption and vitamin D hormone production by mammalian kidney, we have devoted our efforts to the study of a mutant mouse model (Hyp). Studies from our laboratory have demonstrated that Na(+)-dependent phosphate transport is significantly reduced in renal brush border membrane vesicles derived from Hyp mice and that the regulation of the renal mitochondrial enzymes which metabolize 25-hydroxyvitamin D3 (25-OH-D3) is impaired in the mutant strain. The demonstration of abnormal phosphate transport and 25-OH-D3 metabolism in proximal tubule cells derived from Hyp kidney after 6-8 days in culture indicates that the mutant renal phenotype is independent of circulating factors and, therefore, intrinsic to the kidney. However, the precise relationship between these two proximal tubular abnormalities is poorly understood. Because the Hyp mutation segregates as a Mendelian trait, it is very likely that one mutant gene is responsible for the biochemical and clinical phenotype. Several hypotheses are put forth to explain the nature of the primary mutation in the Hyp mouse.

Colchicine blocks the action of parathyroid hormone but not nicotinamide on renal phosphate transport

Nicotinamide, like parathyroid hormone, is a rapidly acting specific inhibitor of Na +-dependent transport of phosphate (P i) across the brush-border membrane of the proximal tubule of the mammalian kidney. Pretreatment of rats with colchicine (0.7 mg/kg body weight) for 1 h led to a significantly diminished phosphaturic response to parathyroid hormone (synthetic 1–34 fragment, 4 μ/kg). In contrast, the same dose of colchicine had no effect on the renal response to nicotinamide (1.0 g/kg), measured both as the change in urinary P i excretion and as Na +-dependent P i uptake by isolated brush-border membrane vesicles. These data suggest indirectly that the intracellular mechanism that mediates the inhibitory effects of nicotinamide on renal P i transport does not require intact microtubules.

Phorbol esters inhibit phosphate uptake in opossum kidney cells: A model of proximal renal tubular cells

The effects of phorbol esters and diacylglycerol on phosphate uptake in opossum kidney (OK) cells were investigated to assess the possible role of Ca 2+-activated, phospholipid dependent protein kinase (protein kinase C) on renal phosphate handling. OK cells are widely used as a model of proximal renal tubular cells and are reported to possess a Na +-dependent phosphate transport system. Phorbol-12,13-dibutyrate (PDBu) inhibited phosphate uptake. This inhibitory effect was synergistically enhanced with A23187. 4β-phorbol 12,13-didecanoate inhibited phosphate uptake, while 4α-phorbol 12,13-didecanoate did not. 1-oleoyl-2-acetyl-glycerol (OAG), a synthetic diacylglycerol, also exhibited an inhibitory effect on phosphate uptake. These data suggest the possible involvement of protein kinase C in proximal renal tubular phosphate transport.

Possible involvement of inositol phosphates and calmodulin in calcitonin-induced stimulation of phosphate transport in LLC-PK 1 cells

The present study investigated the possible involvement of phosphatidylinositol breakdown and Ca2+-calmodulin complex in the calcitonin-induced stimulation of phosphate transport in LLC-PK1 cells. This cell line with calcitonin receptors possesses Na+-dependent phosphate transport and has been employed as a model for studying the mechanism of renal tubular phosphate transport. (Asu1,7) eel calcitonin stimulated the phosphate transport in LLC-PK1 cells in a dose-dependent fashion with accompanying increase of inositol triphosphate (IP3) production. When the cells were preincubated with the potent calmodulin antagonist, w-7 or w-13, the stimulatory effect of calcitonin on phosphate transport was significantly inhibited. These findings indicate that Ca2+-calmodulin complex formed by increased cytosolic Ca2+, which is mobilized from intracellular pools by IP3, may be involved in the signal transduction of calcitonin in these cells.

Effect of pH on the kinetics of Na +-dependent phosphate transport in rat renal brush-border membranes

The kinetics of Na +-dependent phosphate uptake in rat renal brush-border membrane vesicles were studied under zero-trans conditions at 37°C and the effect of pH on the kinetic parameters was determined. When the pH was lowered it turned out to be increasingly difficult to estimate initial rates of phosphate uptake due to an increase in aspecific binding of phosphate to the brush border membrane. When EDTA or β-glycerophosphate was added to the uptake medium this aspecific binding was markedly reduced. At pH 6.8, initial rates of phosphate uptake were measured between 0.01 and 3.0 mM phosphate in the presence of 100 mM Na +. Kinetic analysis resulted in a non-linear Eadie-Hofstee plot, compatible with two modes of transport: one major low-affinity system ( K m ≈ 1.3 mM), high-capacity system ( V max ≈ 1.1 nmol/s per mg protein) and one minor high-affinity ( K m ≈ 0.03 mM), low-capacity system ( V max ≈ 0.04 nmol/s per mg protein). Na +-dependent phosphate uptake studied far from initial rate conditions i.e. at 15 s, frequently observed in the literature, led to a dramatic decrease in the V max of the low-affinity system. When both the extra- and intravesicular pH were increased from 6.2 to 8.5, the K m value of the low-affinity system increased, but when divalent phosphate is considered to be the sole substrate for the low-affinity system then the K m value is no longer pH dependent. In contrast, the K m value of the high-affinity system was not influenced by pH but the V max decreased dramatically when the pH is lowered from 8.5 to 6.2. These results suggest that the low-affinity, high-capacity system transports divalent phosphate only while the high-affinity, low-capacity system may transport univalent as well as divalent phosphate. Raising medium sodium concentration from 100 to 250 mM increased Na +-dependent phosphate uptake significantly but the pH dependence of the phosphate transport was not influenced. This observation makes it rather unlikely that pH changes only affect the Na + site of the Na +-dependent phosphate transport system.

Intestinal phosphate and calcium absorption: joint regulation by thyroid hormones and 1,25-dihydroxyvitamin D3.

Thyroxine (T4) and triiodothyronine (T3) activate Na+-dependent inorganic phosphate (Pi) transport in organ-cultured embryonic chick small intestine. Induction of transport activity requires intact protein synthesis and can be expressed in enterocytes with varying degrees of differentiation. T3 and T4 exert their effect independent of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), which is shown to stimulate Pi uptake only in the final stage of embryonic differentiation. At this time point, a potentiating effect of 1,25(OH)2D3 and T4 on Pi transport in cultured jejunum can be demonstrated. Thyroid hormones appear to stimulate Na+ gradient-driven Pi transport without concomitantly raising (Na+-K+)-ATPase activity. T4 has no influence whatsoever on calcium uptake by cultured embryonic small intestine while 1,25(OH)2D3 is effective at all stages of embryonic development investigated (day 15-20). However, when both hormones were present in the culture medium, the effect of 1,25(OH)2D3 on calcium transport is doubled. Our results suggest that the hyperphosphataemia associated with hyperthyroidism is likely to result, at least in part, from the independent effect of thyroid hormones as well as from their potentiation of the 1,25(OH)2D3 action on Na+-dependent intestinal Pi transport. In addition, their permissive effect on 1,25(OH)2D3-induced calcium absorption provides an explanation for unaltered calcium absorption in a number of hyperthyroid patients, although reduced plasma levels of 1,25(OH)2D3 are generally observed in this condition.

Regulation of sodium-coupled phosphate transport by extracellular phosphate in cultured kidney cells (JTC-12).

Cultured monkey kidney cells (JTC-12) have a Na+-dependent phosphate (Pi) transport system with characteristics similar to that of the renal proximal tubule. Na+-dependent Pi uptake in JTC-12 cells is affected by Pi concentrations in the culture medium. In this investigation, further characterization of this phenomenon was carried out. Lowering the concentration of extracellular Pi (3.0 mmol/l to 0.3 mmol/l) induced an increase in Na+-dependent Pi uptake compared with that in control cells maintained in 3.0 mmol/l Pi, whereas Na+-dependent transport of hexose and amino acid was not altered. This response was first evident at 4 h after the extracellular Pi concentration was reduced and slowly developed over the subsequent 24 h. Kinetic analysis showed an increase in the Vmax without a change in the apparent Km for Pi in cells cultured in the low Pi concentration compared with control cells. The response of Pi uptake was only partially prevented by cycloheximide, suggesting that both protein synthesis-dependent and -independent mechanisms are involved in the development of the response. Insulin, which has a stimulatory effect on Pi uptake in JTC-12 cells, did not affect this response. These data indicate that JTC-12 cells respond to changes in extracellular Pi concentration by changing the Na+-dependent Pi uptake system. This response has a number of properties typical of the phenomenon of adaptation of renal Pi transport in vivo to dietary phosphorus load.

Parathyroid hormone inhibits phosphate transport in OK cells but not in LLC-PK1 and JTC-12.P3 cells.

Na+-dependent phosphate transport and its response to parathyroid hormone (PTH) has been investigated in three continuous cell lines of renal epithelial origin (LLC-PK1, JTC-12.P3, and OK). The apparent Km for phosphate was similar, but the maximal transport rate (Vmax) was markedly different in the three cell lines. PTH and forskolin produced an increase of cellular adenosine 3',5'-cyclic monophosphate (cAMP) in all cell lines, but Na+-dependent phosphate transport was inhibited exclusively in the OK cells (a threefold reduction of influx after 4 h of exposure to 10(-10) M PTH). The change in phosphate transport is accounted for by a lowered Vmax (30.8 +/- 5.3 vs. 10.2 +/- 1.1 pmol X mg-1 X 3 min-1). The reduction in phosphate transport was reversible, such that 5 h after removal of PTH the Vmax had increased threefold over the inhibited state. Addition of PTH did not alter Na+-dependent L-alanine influx in the OK cells. Experiments with apical membrane vesicles showed that the change in Vmax occurred at the membrane level. It is concluded that the regulatory event responsible for PTH-reduced phosphate transport is beyond cAMP. Of the cell lines studied, only OK cells have a complete regulatory cascade.

Phosphonocarboxylic acids as specific inhibitors of Na+-dependent transport of phosphate across renal brush border membrane.

We investigated interactions of phosphonoformic acid (PFA), phosphonoacetic acid (PAA), and other phosphonyl derivatives with the Na+ gradient [Na+ extravesicular greater than Na+ intravesicular; Nao+ greater than Na+i]-dependent transport system for phosphate (Pi) in renal cortical brush border membrane vesicles (BBMV). PFA and PAA inhibited in a dose-dependent manner the Na+ gradient [Na+o greater than Na+i]-dependent uptake of Pi by rat kidney BBMV. PFA was a more potent inhibitor than PAA while phosphonopropionic acid, hydroxymethylphosphonic acid, and phenylphosphonic acid had no effect on Pi transport. The inhibitory effect of PFA was competitive (Ki approximately equal to 4.6 X 10(-4) M) and reversible upon dilution. The uptake of Pi by BBMV in the absence of Na+ gradient [Nao+ = Na+i] was also inhibited by PFA. The PFA had no effect on uptake of L-[3H]proline, D-[3H]glucose, or 22Na+ by BBMV nor did it alter intravesicular volume of BBMV. The relative (%) extent of inhibition by PFA was not altered by changes in the extravesicular pH or changes in the steepness of the Na+ gradient [Nao+ greater than Na+i]. The inhibition of PFA was analogous in renal BBMV from rats, mice, rabbits, or dogs. Unlike other known inhibitors of brush border membrane (BBM) transport of Pi, e.g. arsenate, NAD, and ethane-1-hydroxy-1,1-diphosphonate, PFA and PAA had no inhibitory effect on BBM-bound or solubilized alkaline phosphatase. Also, PFA did not interfere with the activity of renal cortical (Na-K)ATPase. Administration of PFA (0.5 g/kg/day, intraperitoneally) to thyroparathyroidectomized rats fed a low Pi diet elicited an increase in urinary excretion of Pi, but did not change the excretion of Na+, K, and Ca2+. The results show that the PFA, and to a lesser degree PAA, are specific competitive inhibitors of the Na+-Pi cotransport in renal cortical BBM and are suitable probes for studies of this transport system.

Increased transport of inorganic phosphate in renal brush borders of spontaneously hypertensive rats.

Phosphate metabolism was studied in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) by measuring serum phosphate concentrations and 24-h urinary phosphate excretions in rats placed in metabolic cages from 6 to 23 wk of age. Serum phosphate concentrations in SHR were significantly lower than those in WKY at 6, 12, and 20 wk of age. In addition, 24-h urinary phosphate excretion was lower in SHR relative to WKY from 6 through 23 wk of age. The hypophosphaturia in SHR was accompanied with an increase in the maximal transport rate of Na+-dependent phosphate transport in renal brush border membrane vesicles (BBMV) isolated from kidney cortex at 6, 12, and 20 wk of age. The apparent affinity for phosphate did not differ significantly between WKY and SHR at all ages studied. A direct relationship between maximal Na+-dependent phosphate transport rates in BBMV and serum phosphate concentrations was observed in both strains. In SHR, phosphate homeostasis is disturbed from 6 wk of age on.

Effect of the Hyp mutation and diet-induced hyperparathyroidism on renal parathyroid hormone- and forskolin-stimulated adenosine 3',5'-monophosphate production and brush border membrane phosphate transport.

The present study was undertaken to investigate the effect of the Hyp mutation and diet-induced hyperparathyroidism on renal responsiveness to PTH and forskolin and to determine whether the renal brush border membrane phosphate transport defect is expressed in the vitamin D- and calcium-deficient Hyp mouse. Our results indicate that PTH is a potent activator of cAMP synthesis in renal slices obtained from vitamin D-replete normal mice and Hyp littermates. However, in the mutants, the amount of cAMP produced in response to similar concentrations of PTH (0.005-5 U/ml) is 55% of normal (P = 0.0076). PTH-dependent cAMP synthesis is significantly reduced in both normal and Hyp mice fed the vitamin D-deficient low calcium diet, and under these conditions, genotype differences are no longer apparent. In contrast, forskolin-stimulated cAMP production is identical in vitamin D-replete normal and mutant mice. Furthermore, cAMP accumulation in response to forskolin is not decreased by diet-induced hyperparathyroidism in either genotype. Vitamin D and calcium deprivation results in a significant decrease in renal brush border membrane Na+-dependent phosphate transport in both genotypes, and under these conditions, the phosphate transport defect persists in Hyp mice. Finally, vitamin D and calcium deprivation has no effect on renal brush border membrane alkaline phosphatase activity. The present results suggest that the catalytic subunit of adenylate cyclase is intact in the mutant strain and that the blunted renal response to PTH in vitamin D-replete Hyp mice as well as in vitamin D- and calcium-deficient mice may be due to uncoupling of the PTH-receptor-adenylate cyclase system. The data also suggest that the expression of the renal phosphate transport defect in Hyp mice is independent of PTH status and alkaline phosphatase activity.

Mechanisms of ion transport regulation by parathyroid hormone: cAMP/Ca2+/calmodulin and phospholipid dependent phosphorylation.

Since 1976, when the function of the Na+ dependent phosphate symport of the renal brush border membrane (BBM) was first well described, numerous regulatory mechanisms of renal phosphate transport have been shown to exert effects on this carrier protein similar to their affect on proximal tubular phosphate transport (1,2). Parathyroid hormone (PTH), which decreases renal phosphate reabsorption, also causes a decrease in Na+ dependent phosphate transport in isolated brush border membrane vesicles (BBMV) (3,4). This effect is exerted as a decrease in the Vmax of the carrier activity compatible with a mechanism acting through a decrease in the number of membrane functional units or a decrease in the overall activity of existing units. The mechanism of action of PTH has long been thought to occur through stimulation of adenylate cyclase, increasing c-AMP, and activation of a c-AMP dependent protein kinase (5,6).

Cellular mechanisms of inorganic phosphate transport in kidney.

articleCellular mechanisms of inorganic phosphate transport in kidneyP. Gmaj, and H. MurerP. GmajSearch for more papers by this author, and H. MurerSearch for more papers by this authorPublished Online:01 Jan 1986https://doi.org/10.1152/physrev.1986.66.1.36MoreSectionsPDF (6 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByDisorders of the Endocrine SystemEthyl Chloride18 June 2004Phosphate absorption in Aplysiacalifornica gut: glucocorticoid inhibitionCanadian Journal of Physiology and Pharmacology, Vol. 81, No. 4Regulation of Na/Pi Transporter in the Proximal TubuleAnnual Review of Physiology, Vol. 65, No. 1pH dependence of Na+/myo-inositol cotransporters in rat thick limb cellsKidney International, Vol. 62, No. 6Thyroid-hormone-induced phosphate absorption in Aplysia californica gut is mediated through protein synthesisCanadian Journal of Physiology and Pharmacology, Vol. 80, No. 12Basolateral Phosphate Transport in Renal Proximal-Tubule-Like OK Cells 129 November 2016 | Experimental Biology and Medicine, Vol. 227, No. 8Sodium-Phosphate Symport by Aplysia Californica GutZoological Science, Vol. 19, No. 2Renal and Skeletal Actions of Parathyroid Hormone (PTH) and PTH-Related ProteinAmino acids involved in sodium interaction of murine type II Na + ‐P i cotransporters expressed in Xenopus oocytes1 March 2001 | The Journal of Physiology, Vol. 531, No. 2Evolution of the Na-Pi cotransport systemsAndreas Werner, and Rolf K. H. Kinne1 February 2001 | American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, Vol. 280, No. 2PLC-γ1 Signaling Pathway and Villin Activation Are Involved In Actin Cytoskeleton Reorganization Induced by Na+/Pi Cotransport Up-regulation1 April 2000 | Molecular Medicine, Vol. 6, No. 4Proximal Tubular Phosphate Reabsorption: Molecular MechanismsHeini Murer, Nati Hernando, Ian Forster, and Jürg Biber10 January 2000 | Physiological Reviews, Vol. 80, No. 4Adaptation to changes in dietary phosphorus intake in health and in renal failureJournal of Laboratory and Clinical Medicine, Vol. 129, No. 2Immunodetection and characterization of proteins implicated in renal sodium/phosphate cotransportBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1190, No. 2Inhibition by cyclic AMP and phorbol esters of sodium-dependent uptake of phosphate by rat hepatocytesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1190, No. 2Expression of Na/Pi cotransport from opossum kidney cells in Xenopus laevis oocytesBiochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1178, No. 2Sodium-dependent transport of phosphate by rat liver plasma membrane vesiclesBiochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1143, No. 2Uptake of phosphate by rat hepatocytes in primary culture: a sodium-dependent system that is stimulated by insulinBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1148, No. 1Expression of rat renal sodium/phosphate contransporter in Xenopus laevis oocytesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1104, No. 1Molecular size of the renal sodium/phosphate symporter in native and reconstituted systemsBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1104, No. 1Inhibition of sodium-phosphate cotransport in renal brush-border membranes with the stilbenedisulfonate, H2-DIDSBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1069, No. 1Some characteristics of sodium-independent phosphate transport across renal basolateral membranesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1064, No. 2The stepwise hydrolysis of adenine nucleotides by ectoenzymes of rat renal brush-border membranesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1030, No. 1Different molecular sizes for Na+-dependent phosphonoformic acid binding and phosphate transport in renal brush border membrane vesiclesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1028, No. 2Neomycin inhibits PDGF-induced IP3 formation and DNA synthesis but not PDGF-stimulated uptake of inorganic phosphate in C3H/10T1/2 fibroblastsBiochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1054, No. 2Effect of parathyroid hormone and dietary phosphate on phosphate transport in renal outer cortical and outer medullary brush-border membrane vesiclesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1024, No. 1Increase in membrane fluidity modulates sodium-coupled uptakes and cyclic AMP synthesis by renal proximal tubular cells in primary cultureBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1022, No. 1Thiol redox and phosphate transport in renal brush-border membrane. Effect of nicotinamideBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1021, No. 1[29] Phosphate transport in established renal epithelial cell lines[37] Stimulus-secretion coupling: General models and specific aspects in epithelial cellsMechanisms of mammary gland ion transporComparative Biochemistry and Physiology Part A: Physiology, Vol. 97, No. 3Effect of phosphonoformic acid, dietary phosphate and the Hyp mutation on kinetically distinct phosphate transport processes in mouse kidneyBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 984, No. 2The Na+/Pi-cotransporter of OK cells: reaction and tentative identification with N-acetylimidazoleBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 981, No. 2[16] Cation-anion cotransportTransport of sulfate and phosphate in small intestine and renal proximal tubule: Methods and basic propertiesComparative Biochemistry and Physiology Part A: Physiology, Vol. 90, No. 4Colchicine blocks the action of parathyroid hormone but not nicotinamide on renal phosphate transportBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 905, No. 2Phosphate homeostasisMolecular Aspects of Medicine, Vol. 9, No. 4 More from this issue > Volume 66Issue 1January 1986Pages 36-70 Copyright & PermissionsCopyright © 1986 the American Physiological Societyhttps://doi.org/10.1152/physrev.1986.66.1.36PubMed3003769History Published online 1 January 1986 Published in print 1 January 1986 Metrics

Actions of NAD+ on renal brush border transport of phosphate in vivo and in vitro.

Previous studies showed that an increase in NAD+ content in renal cortex in vivo was accompanied by specific inhibition of Na+-dependent inorganic phosphate (Pi) transport across the renal brush border membrane (BBM). Further, in vitro addition of NAD+ to isolated renal BBM vesicles specifically inhibited Na+ gradient-dependent transport of Pi. The present study examined some aspects of the mechanism of this inhibition by NAD+ in vitro and in vivo. When NAD+ was increased in vivo by nicotinamide injection, the apparent Vmax was decreased, but the apparent Km was not different, indicating apparent noncompetitive inhibition. In the presence of 0.3 mM NAD+ added in vitro, the apparent Km for Na+-dependent Pi transport by BBM vesicles was increased, whereas the apparent Vmax was unchanged, indicating apparent competitive inhibition. These changes in apparent Km and apparent Vmax were identical when Pi uptake was measured either at 30-s or at 5-s (the initial rate) incubation times. Inhibition of Pi transport by BBM vesicles in vitro was due primarily to the action of intact added NAD+, although there may be some contribution by isotope dilution due to Pi released from NAD+ by enzymatic hydrolysis. Although in vitro inhibition of Pi transport by added NAD+ was reversed by washing the BBM, the inhibition due to increased NAD+ in vivo persisted after extensive washing of the isolated BBM. The specificity of the inhibitory effect of NAD+ in vivo was indicated by the finding that changes in renal cortical content of ATP or Pi, evoked by loading with glycerol or fructose, did not change BBM transport of Pi.(ABSTRACT TRUNCATED AT 250 WORDS)

Diuretic effects on renal brush border membrane transport and metabolism

Previous studies have indicated that the thiazide diuretics exert effects on proximal electrolyte transport. To determine whether the locus of these effects is at the brush border membrane (BBM) and if renal metabolism is affected, adult female Sprague-Dawley rats were acutely treated with either 1 mg/kg metolazone, 20 mg/kg chlorothiazide followed by a 20 mg/kg/hr maintenance infusion, 10 mg/kg acetazolamide followed by a 10 mg/kg/hr maintenance infusion, or the vehicles only. Administration of these agents resulted in an approximately tenfold increase in sodium excretion. Neither urinary phosphate nor inulin excretion changed significantly in any group. Sodium dependent BBM vesicle phosphate transport was examined at 0.15, 0.5, and 1 and 120 minute incubation periods in the diuretic treated groups and their respective control groups. Decreased uptake was seen in all pre-equilibrium time points in rats treated with metolazone: 0.15 minutes: 221 ± 24 pmoles/mg protein (pmol/mg prot) in control rats versus (vs) 185 ± 23 pmoles/mg prot in metolazone-treated animals (P < .05); 0.5 minutes: 463 ± 54 vs 369 ± 49 pmol/mg prot (P < .005); 1 minute: 549 ± 74 vs 460 ± 61 pmol/mg prot (P < .05); no significant difference in phosphate transport was noted at the two hour equilibrium time point. No significant differences in sodium dependent phosphate transport existed between chlorothiazide or acetazolamide treated rats and control animals. Substrate-stimulated renal gluconeogenesis did not differ between metolazone treated and control animals. We therefore conclude that metolazone inhibits phosphate transport through an effect on the BBM and does not affect renal gluconeogenesis in the rat.

Effects of 1,25-dihydroxycholecalciferol on phosphate transport in vitamin D-deprived rats.

To clarify the role of vitamin D in renal phosphate transport, weanling rats were fed a vitamin D-deficient diet containing 1.8% calcium and 1.2% phosphorus. After 5-6 wk, the rats were normocalcemic, normophosphatemic, and had normal levels of PTH. Assays of vitamin D metabolites revealed undetectable plasma levels of 25(OH)D, and 1,25(OH)2D levels of 92 +/- 16 pg/ml in partially vitamin D-depleted (PVDD) rats and 169 +/- 58 pg/ml in normal rats. PVDD rats had increased phosphate excretion, both absolute and fractional, and a decrease in Na+ gradient-dependent Pi transport in proximal tubular brush border membrane vesicles (BBMV) prepared from their kidneys. Vitamin D repletion of PVDD rats with 1,25(OH)2D3, 15 pmol/100 g body wt, decreased fractional excretion of Pi from 22.6 +/- 1.9 to 13.5 +/- 1.3%; the latter values were similar to normal rats. Repletion with 1,25(OH)2D3 also increased Na+-dependent phosphate transport in BBMV from 322 +/- 24 pmol X mg protein-1 X 15 s-1 in BBMV from PVDD rats to 698 +/- 70 pmol X mg protein-1 X 15 s-1. Repletion with larger doses of 1,25(OH)2D3 produced hypercalcemia and hyperphosphatemia from intestinal absorption, an increase in phosphate excretion, and a blunted response of Pi transport to 1,25(OH)2D3. Prevention of hyperphosphatemia by dietary adjustments allowed full expression of the stimulatory effects of 1,25(OH)2D3 on Pi transport. These later data may partially explain the inhibitory effects reported in prior studies in which plasma Pi was not controlled and the larger doses of 1,25(OH)2D3 administered.(ABSTRACT TRUNCATED AT 250 WORDS)

Divalent metal is required for both phosphate transport and phosphate binding to phosphorin, a proteolipid isolated from brush-border membrane vesicles.

The Na+-dependent phosphate transport system in the brush border of rabbit kidney exhibits a positive requirement for a divalent metal ion. Treatment of the brush-border membrane vesicles (BBMV) with a divalent metal chelator in combination with the divalent metal ionophore A23187 dramatically and selectively decreased the Na+-dependent uptake of phosphate; Na+-independent uptake of phosphate was not affected. The combination of chelator plus A23187 also inhibited uptake of phosphate in the presence of Na+ but in the absence of a gradient for sodium across the BBMV. This indicates that the inhibitor is not a result of an alteration in the Na+ gradient by chelator plus ionophore. The inhibited Na+ gradient-dependent transport of phosphate was restored by removing the chelator and adding Mn2+ to the BBMV. The phosphate-binding proteolipid (phosphorin) isolated from rabbit kidney BBMV binds inorganic phosphate with high affinity and specificity. Binding of phosphate to phosphorin is also inhibited by divalent metal chelators and can be restored by addition of a divalent metal. We conclude that a divalent metal ion is required both for the Na+-dependent phosphate transport in BBMV and for the binding of phosphate to the proteolipid phosphorin. These findings are consistent with our suggestion that phosphorin is a component of the Na+-dependent phosphate transport system in renal brush-border membranes.