Pi Cotransporter Research Articles

Renal reabsorption of phosphate during development: transport kinetics in BBMV

The mechanism responsible for enhanced reabsorption of phosphate (Pi) in growing animals was assessed in renal brush-border membrane vesicles (BBMV) prepared from 3- to 14-day-old and greater than 57-day-old guinea pigs. On standard diet, Vmax (pmol.mg-1.s-1) of Na+-Pi cotransport was higher (P less than 0.001) in newborn (650 +/- 77) than in adult (144 +/- 17) but Vmax of Na+-glucose cotransport did not differ with age. Low dietary Pi did not affect significantly Vmax of Na+-Pi cotransport in the newborn (P greater than 0.8) but increased it in the adult (to 318 +/- 32, P less than 0.05). A high Pi intake resulted in a smaller relative decrease in Vmax in the newborn than in the adult (27 vs. 44%, P less than 0.05). In the newborn, the serum Pi (mM) decreased on a low-Pi diet (from 1.8 +/- 0.1 to 0.8 +/- 0.1, P less than 0.001) and rose by twofold on the high-Pi diet (to 3.5 +/- 0.2, P less than 0.001). In the adult, there were no significant changes in serum Pi with changes in Pi intake (P greater than 0.5). Thus in the newborn the Na+-Pi cotransport system is characterized by high transport capacity but low adaptability to changes in dietary Pi.

Different mechanisms of adaptive increase in Na+-Pi cotransport across renal brush-border membrane

We explored the biochemical mechanism by which thyroid hormone (T3) and low-phosphate diet (LPD) cause an adaptive increase in Na+-Pi cotransport across renal brush-border membrane (BBM). The rate of Na+-Pi cotransport was determined by 32Pi uptake by BBM vesicles (BBMV), and the number of Na+-Pi symporters was assessed by binding of [14C]phosphonoformic acid (PFA) on BBMV. In BBMV of both T3-treated rats and LPD-fed rats, the Na+ gradient-dependent 32Pi uptake increased (Vmax increased; Km Pi was not changed). The Na+-dependent [14C]PFA binding on BBMV increased (higher Vmax, no change in Km PFA) in response to T3, but it remained unchanged in rats fed LPD. Both the increase of Na+-Pi cotransport and of Na+-dependent [14C]PFA binding in response to T3 were blocked by actinomycin D or cycloheximide. Addition of benzyl alcohol to BBMV in vitro increased Na+-Pi cotransport, but [14C]PFA binding did not change; the [3H]phlorizin binding and cotransports of other solutes decreased or did not change. The exposure of BBMV to cholesterol decreased Na+-Pi cotransport without changing [14C]PFA binding. We suggest that the adaptive increase of Na+-Pi cotransport elicited by T3 is due to an increase in number of Na+-Pi cotransporters in BBM. In contrast, in response to LPD the number of Na+-Pi cotransporters is unchanged, and the increased Na+-Pi cotransport is due to faster translocation of Na+ with Pi due to enhanced fluidity of BBM.

Identification of Na+,Pi-binding protein in kidney and intestinal brush-border membranes.

An Na+, Pi-binding protein has been extracted from kidney and intestinal brush-border membranes with an organic solvent and has been purified by Kieselghur and Sephadex LH-60 chromatography. The molecular mass of this protein has been estimated to be about 155 kDa as determined by gel-filtration chromatography on Sepharose 2B. Under denaturing conditions, polyacrylamide-gel electrophoresis revealed a monomer of molecular mass about 70 kDa. The protein has high specificity and high affinity for Pi [K0.5 (concentration at which half-maximal binding is observed) near 10 microM]. Na2+ binding also exhibits saturation behaviour, with a K0.5 near 7.5 mM. Pi binding is inhibited by known inhibitors of Pi transport in brush-border membrane vesicles. It appears that this protein could be involved in Na+/Pi co-transport across the renal and intestinal brush-border membranes.

Modulation of Na+-Pi cotransport in opossum kidney cells by extracellular phosphate.

The effect of the extracellular concentration of Pi on the Na+-dependent phosphate transport activity of OK cells was investigated. When incubated with extracellular Pi at concentrations of 200 microM or less, Na+-Pi cotransport increased approximately twofold in OK cells compared with control cells (kept in 0.85 mM Pi), whereas other Na+-dependent transport activities were not affected. After Pi deprivation, Na+-Pi cotransport could be inhibited to a similar extent (80%) by parathyroid hormone (PTH) as in control cells, suggesting that the PTH-sensitive Na+-Pi cotransport activity is also regulated by extracellular Pi. The increase of Na+-Pi cotransport was maximally expressed after 6 h and could be prevented by cycloheximide (70 microM) but not by actinomycin D (0.5-5 g/ml). However, the adaptive response was completely blocked by 3'-deoxyadenosine (cordycepin) at 100 microM. From these data, it is concluded that the upregulation of Na+-Pi cotransport in OK cells due to low extracellular Pi is controlled at a posttranscriptional level.

α-Cl-α-Br-phosphonoacetic acid is a potent and selective inhibitor of Na +/Pi cotransport across renal cortical brush border membrane

We found that alpha-Cl-alpha-Br-phosphonoacetate (ClBrPAA) is a competitive, solute-specific inhibitor of Na+/Pi cotransport across renal cortical brush border membrane. Inhibition by ClBrPAA (Ki = 62 microM) is more than three times more effective than inhibition by phosphonoformate (PFA), the most potent Na+/Pi cotransport inhibitor known to date, and 26 times more effective than the parent compound, phosphonoacetate (PAA). These observations indicate that substitution of bromine and chlorine atoms at the alpha-carbon of PAA greatly enhances its efficacy as a competitive inhibitor of Na+/Pi cotransport. As ClBrPAA is much less inhibitory than PAA and PFA towards viral DNA polymerases and did not inhibit human alpha-DNA polymerase (ref. 10), the results also demonstrate that Na+/Pi cotransport inhibition can be dissociated from inhibition of DNA polymerases by phosphonocarboxylate compounds.

Adjustment to Pj depletion of the Na+—Pi co-transporter of mouse proximal tubule cells

Conference Article| June 01 1988 Adjustment to Pj depletion of the Na+—Pi co-transporter of mouse proximal tubule cells PETER J. BUTTERWORTH; PETER J. BUTTERWORTH 1Department of Biochemistry, King's College (London), Kensington Campus, Campden Hill Road, London W8 7AH, U.K. Search for other works by this author on: This Site PubMed Google Scholar MUSSARAT JAHAN MUSSARAT JAHAN 1Department of Biochemistry, King's College (London), Kensington Campus, Campden Hill Road, London W8 7AH, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1988) 16 (3): 353. https://doi.org/10.1042/bst0160353 Article history Received: November 13 1987 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation PETER J. BUTTERWORTH, MUSSARAT JAHAN; Adjustment to Pj depletion of the Na+—Pi co-transporter of mouse proximal tubule cells. Biochem Soc Trans 1 June 1988; 16 (3): 353. doi: https://doi.org/10.1042/bst0160353 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1988 Biochemical Society1988 Article PDF first page preview Close Modal You do not currently have access to this content.

Study of the mechanism by which the Na+-Pi co-transporter of mouse kidney proximal-tubule cells adjusts to phosphate depletion.

1. Proximal-tubule cells isolated from mouse kidney after digestion with collagenase take up Pi by an Na+-dependent and saturable process mediated by the Na+-Pi co-transporter of the brush-border membrane. 2. Pi depletion of the cells is accompanied by a stimulation of Pi-transport activity. Kinetic investigations reveal that Vmax. is increased by 90% and Km decreased by 50% after Pi depletion. Transport activity returns to normal values after incubation for 30 min at 37 degrees C of Pi-depleted cells in normal medium containing 1 mM-Pi, but the fall in transport activity under these conditions is inhibited by colchicine. 3. The energy of activation of Na+-Pi co-transport activity of depleted cells differs greatly from that found for normal replete cells. 4. The results provide evidence that stimulation of transport by Pi depletion arises from an increase in the number of carrier sites in the brush-border membrane. Additionally, changes in the properties of the transporter occur which may reflect altered phospholipid-carrier-protein interaction in the Pi-depleted condition.

Interactions of [14C]phosphonoformic acid with renal cortical brush-border membranes. Relationship to the Na+-phosphate co-transporter.

Since phosphonoformic acid (PFA) acts as a specific competitive inhibitor of Na+-Pi co-transport across renal brush-border membrane (BBM), we employed the [14C]PFA as a probe to determine the mechanism of its interaction with rat renal BBM. The binding of [14C]PFA to BBM vesicles (BBMV), with Na+ present in extravesicular medium (Na+o), was time- and temperature-dependent. The replacement of Na+o with other monovalent cations reduced the PFA binding by -80%. Cl- was the most effective accompanying monovalent anion as NaCl for maximum PFA binding. The Na+o increased the apparent affinity of BBMV for [14C]PFA binding, but it did not change the maximum binding capacity. The maximum [14C]PFA binding was achieved at Na+o approximately equal to 50 mM. The extent of Na+-dependent [14C]PFA binding correlated (r = 0.98; p less than 0.01) with percent inhibition by an equimolar dose of PFA of the (Na+o greater than Na+i)-dependent BBMV uptake of 32Pi. Intravesicular Na+ (Na+i) decreased [14C]PFA binding, on BBMV, and this inhibition by Na+i was dependent on the presence of Na+o. The increase in Na+i, at constant [Na+]o, decreased the Vmax, but not the Km, for [14C]PFA binding on BBMV. Bound [14C]PFA was displaced from BBMV by phosphonocarboxylic acids proportionally (r = 0.99; p less than 0.05) to their ability to inhibit (Na+o greater than Na+i)-gradient-dependent Pi transport, whereas other monophosphonates, diphosphonates, L-proline, or D-glucose did not influence the [14C]PFA binding. The Na+-dependent binding of [14C]PFA and of [3H]phlorizin by BBMV was 10 times higher than binding of these ligands to renal basolateral membranes and to mitochondria. [14C]PFA probably binds onto the same locus on the luminal surface of BBM, where Pi and Na+ form a ternary complex with the Na+-Pi co-transporter.

Calcitonin inhibits Na+ gradient-dependent phosphate uptake across renal brush-border membranes.

This study was undertaken to determine whether calcitonin inhibits Na+-(Pi) cotransport across luminal brush-border membrane (BBM) of proximal tubules. Further, we determined the relative inhibitions of inorganic phosphate (Pi) transport in BBM vesicles (BBMV) derived from superficial cortical tissue (BBMV-SC) and juxtamedullary tissue (BBMV-JM). The effects of maximally phosphaturic doses of calcitonin were compared with those of parathyroid hormone (PTH). Experiments were performed in acutely thyroparathyroidectomized (TPTX) rats fed either normal (NPD, 0.7%) or low (LPD, 0.07%) Pi diets. After measurement of the fractional excretion of phosphate (FEPi) by clearance, the BBMV-SC and BBMV-JM were prepared from kidneys of the same animals and uptakes of 32Pi were determined. Both calcitonin and PTH inhibited BBMV transport of Pi to a greater degree in BBMV-JM than in BBMV-SC, in rats fed NPD or LPD. Kinetic analysis shows that administration of calcitonin resulted in marked decrease of apparent Vmax for Pi without any changes in apparent Km for Pi. However, in spite of a decreased capacity for Na+ gradient-dependent 32Pi uptake in BBMV-JM and much lesser in BBMV-SC in response to administration of calcitonin and PTH in Pi-deprived rats, these phosphaturic peptides did not increase FEPi. We conclude that calcitonin administration decreases the capacity for Na+-Pi cotransport across BBM in proximal tubules of the acutely TPTX rat.

Inhibition of human renal epithelial Na +/Pi cotransport by phosphonoformic acid

Phosphonoformic acid was a potent competitive inhibitor of Na+/Pi cotransport by brush border membrane vesicles isolated from human renal cortex (Ki 31 microM). The Na+/Pi cotransport system in rat renal brush border membrane vesicles was much less sensitive to inhibition (Ki 210 microM). Na+/Pi cotransport by cultured cells derived from human renal cortex also was inhibited by phosphonoformic acid. Both in isolated membranes and cells the inhibition was dose-dependent, competitive, and specific for Pi. The effectiveness of phosphonoformic acid on Na+/Pi cotransport in human kidney suggests that its administration may be a useful maneuver to increase renal Pi excretion.

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.

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

1622 K+ STIMULATED PHOSPHATE (Pi) CO-TRANSPORT IN FETAL LAMB (FL) KIDNEY

In the adult kidney, carrier mediated Pi co-transport is highly Na+ specific. Studies in our laboratory showed that Pi uptake (PiU) in renal brush border membrane vesicles (BBMV) from FL kidneys is also stimulated by Na+. In contrast to the adult, we report here that carrier mediated PiU in FL renal BBMV is also energized by a K+ gradient ([K+]o>[K]i). BBMV from 5 FL kidneys were prepared by CaCl2 precipitation and verified by marker enzyme assays, electron microscopy, and PiU into osmoticaly active vesicles. Peak 60s PiU with a 100mM Na+ and K+ gradient was 1968±65 and 1010±133 pMol/mg BBMV protein, respectively (p<.01). PiU for each was greater than diffusion PiU (p<.001 each). The effect of trans-stimulation was studied by preloading BBMV with 5mM Pi. Initial 12s PiU in preloaded BBMV was 1007±181 pMol/mg BBMV protein and was 4-fold higher than PiU in non-loaded BBMV (p<.001). The effect of extravesicular (O) pH was studied by measuring initial 15s PiU in BBMV with constant intravesicular pH 7.4 and varying O pH from 6.0, 7.4 and 8.0. PiU was 700±64, 1983±230 and 411±10 pMol/mg BBMV protein for OpH 6.0, 7.4 and 8.0 respectively. PiU at pHO=pHi was higher than that for OpH 6.0 and OpH 8.0 (p<.01; p<.001). The results demonstrate K+-Pi symport in FL kidney. The co-transport system may represent shared affinity by K+ with Na+ for the cation site on the carrier or a different carrier system.

Insulin stimulates Pi transport in brush border vesicles from proximal tubular segments.

Induction of hyperinsulinemia in dogs results in enhanced reabsorption of Pi from glomerular filtrate in the renal proximal tubule. To determine whether this may be a direct action of insulin mediated by altered transport characteristics of the proximal tubular brush border membrane, we measured Na+-dependent 32Pi transport in brush border membrane vesicles prepared from isolated proximal tubular segments originating from dog kidney that had been incubated with or without insulin. Specific high affinity binding sites for insulin were detected in proximal tubular segments. Increased initial rates (15 s) of Na+-dependent 32Pi transport were measured in brush border vesicles prepared from segments that had been incubated with insulin. This effect of insulin was concentration dependent over the range of 10(-10) to 10(-6) M insulin. These studies demonstrate the feasibility of using brush border vesicles prepared from proximal tubular segments to study solute transport. Our findings suggest that insulin-induced increased Pi reabsorption in the proximal tubule is mediated by a direct action of insulin on the proximal tubular cell, which results in increased Na+-Pi cotransport across the brush border membrane.

Na+ gradient-dependent Pi uptake in basolateral membrane vesicles from dog kidney.

To ascertain whether Na+ gradient-stimulated 32Pi uptake was demonstrable in renal basolateral membrane vesicles, we measured 32Pi uptake in basolateral membrane suspensions isolated from canine renal cortex and compared solute uptake in basolateral suspensions with that measured in brush border suspensions. Measurements revealed Na+ gradient-dependent 32Pi transport in basolateral preparations. D-[3H] Glucose uptakes in basolateral suspensions were not stimulated by the Na+ gradient in contrast to findings in brush border suspensions. Na+ gradient-dependent 32Pi transport in basolateral suspensions was electrogenic in contrast to that measured in brush border preparations. Unlike 32Pi uptake in brush border preparations, Na+ gradient-dependent 32Pi uptake in basolateral suspensions did not increase as extravesicular pH was increased from 6.5 to 7.5. Na+ gradient-dependent 32Pi uptake in basolateral membranes showed saturation over the range of [Pi] from 5 to 100 microM (apparent Km, 14 +/- 2 microM; apparent Vmax, 34 +/- 2 pmol Pi X mg protein-1 X 30s-1). Our findings are compatible with the presence of an electrogenic Na+-Pi cotransporter in the canine proximal tubular basolateral membrane.

CELLULAR MECHANISM OF RENAL PHOSPHATE (Pi) TRANSPORT BY THE NEWBORN

We have provided evidence that the enhanced renal reabsorption of Pi in growing animals is due to a large extent to an increase in the fractional reabsorption of Pi by the proximal tubule. This prompted us to explore the cellular mechanism responsible for this phenomenon. Vesicles of proximal tubule brush border membranes obtained from guinea pigs of either 1-7 or > 30 days of age were used to assess the kinetics of the Na+ -Pi cotransporter. No significant differences were found between these two age groups either in the Km (0.61 vs 0.75 mM, p > .4) or in the Vmax (1282 vs 1960 pmole/mg protein at 12 sec, p >.2). The Ki, measured in the presence of arsenate in the incubation media, was also found to be similar (3.0 vs. 2.3 mM, p > .2). To assess the Pi concentration gradient across the luminal membrane, we measured intracellular concentration of Pi by spectrophotometry (SP) and by nuclear magnetic resonance (NMR) and found it to be ≃ 3-fold lower in the newborn than in the adult. The exchangeable Pi, measured by NMR, was found to represent only ≃ 25% of the total intracellular Pi, measured by SP (.25 ±.6 vs 1.0±0.10 mM, p <.001 in the newborn and .60 ± .07 vs 2.06 ± .09 mM in the adult, p<.005). In addition, the Na+-independent uptake of Pi by brush border microvesicles was significantly higher in the newborn than in the adult (p<0.05) at concentrations of Pi in the media varying between 0.1 and 4.0 mM. We surmise that, contrary to previous assumptions, a steep downhill Pi concentration gradient exists across the luminal membrane of the proximal tubule and that passive diffusion along this concentration gradient may contribute to the enhanced renal reabsorption of Pi observed in growing animals.

Regulation of Na+-Pi cotransport by 1,25-dihydroxyvitamin D3 in rabbit duodenal brush-border membrane

Brush-border membrane vesicles were isolated from rabbit duodenum by a Mg2+ precipitation method, and phosphate transport was analyzed by a rapid filtration technique. Uptake of inorganic phosphate (Pi) was stimulated by an inwardly directed sodium gradient, indicating the operation of a Na-Pi cotransport system in brush-border membrane vesicles. Treatment of the animals with ethane-1-hydroxy-1,1-diphosphonate (EHDP), which is known to decrease the circulating levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], reduced within 3 days the sodium-dependent Pi transport in the brush-border vesicles. Injections of 1,25(OH)2D3 into rabbits increased within 9 h the sodium-dependent Pi transport in membranes from EHDP-treated animals as well as in untreated ones. The Na-D-glucose cotransport system appeared to be unaffected by these maneuvers. These results suggest that the Na-Pi cotransport system is an important site of regulation of intestinal transepithelial Pi transport by 1,25(OH)2)D3.

Renal brush border membrane adaptation to phosphorus deprivation in the Hyp/Y mouse.

Human X-linked hypophosphataemia (XLH)1 involves a partial loss in net renal reabsorption of phosphate anion (Pi). The mutant male hemizygote has about half the normal renal transport activity2 and this is quite insensitive to circulating parathyroid hormone (PTH)2,3. The renal defect is sufficient to explain persistent hypophosphataemia in XLH. The hypophosphataemic mouse (Hyp)4 also has an X-linked defect in renal reabsorption of Pi (refs 4–7). The mutant male hemizygote (Hyp/Y) has about half the normal renal reabsorption activity and this abnormality persists after parathyroidectomy7. Conservation of the X chromosome during mammalian evolution and evidence for functional homology between species for specific X-linked genes8 have encouraged us to use the Hyp mouse as a model for the renal defect in XLH. We have studied Pi transport in purified renal brush border membrane vesicles (BBMV) prepared from Hyp/Y mice5,9. A selective loss involving almost half the Na+-dependent Pi co-transport activity is present in Hyp/Y membranes. These findings, genetic studies in man2,10 and recent physiological evidence11 suggest that Pi transport in the mammalian nephron occurs by at least two processes: one under the control of an X-linked gene that is more sensitive to PTH inhibition and another process, controlled by an autosomal locus, that is less sensitive to the hormone. We report here on the ability of the Hyp/Y mouse to adapt to phosphorus deprivation; the response is observed both in vivo in net reabsorption of Pi and in vitro in BBMV transport of Pi.