44 results on '"Levi, Moshe"'
Search Results
2. Enhanced phosphate absorption in intestinal epithelial cell-specific NHE3 knockout mice.
- Author
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Xue J, Thomas L, Murali SK, Levi M, Fenton RA, Dominguez Rieg JA, and Rieg T
- Subjects
- Animals, Homeostasis, Intestinal Absorption, Intestinal Mucosa metabolism, Mice, Mice, Knockout, Sodium-Hydrogen Exchanger 3 metabolism, Epithelial Cells metabolism, Phosphates metabolism, Sodium-Hydrogen Exchanger 3 genetics
- Abstract
Aims: The kidneys play a major role in maintaining P
i homeostasis. Patients in later stages of CKD develop hyperphosphatemia. One novel treatment option is tenapanor, an intestinal-specific NHE3 inhibitor. To gain mechanistic insight into the role of intestinal NHE3 in Pi homeostasis, we studied tamoxifen-inducible intestinal epithelial cell-specific NHE3 knockout (NHE3IEC-KO ) mice., Methods: Mice underwent dietary Pi challenges, and hormones as well as urinary/plasma Pi were determined. Intestinal33 P uptake studies were conducted in vivo to compare the effects of tenapanor and NHE3IEC-KO . Ex vivo Pi transport was measured in everted gut sacs and brush border membrane vesicles. Intestinal and renal protein expression of Pi transporters were determined., Results: On the control diet, NHE3IEC-KO mice had similar Pi homeostasis, but a ~25% reduction in FGF23 compared with control mice. Everted gut sacs and brush border membrane vesicles showed enhanced Pi uptake associated with increased Npt2b expression in NHE3IEC-KO mice. Acute oral Pi loading resulted in higher plasma Pi in NHE3IEC-KO mice. Tenapanor inhibited intestinal33 P uptake acutely but then led to hyper-absorption at later time points compared to vehicle. In response to high dietary Pi , plasma Pi and FGF23 increased to higher levels in NHE3IEC-KO mice which was associated with greater Npt2b expression. Reduced renal Npt2c and a trend for reduced Npt2a expression were unable to correct for higher plasma Pi ., Conclusion: Intestinal NHE3 has a significant contribution to Pi homeostasis. In contrast to effects described for tenapanor on Pi homeostasis, NHE3IEC-KO mice show enhanced, rather than reduced, intestinal Pi uptake., (© 2022 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)- Published
- 2022
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3. Mechanisms of phosphate transport.
- Author
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Levi M, Gratton E, Forster IC, Hernando N, Wagner CA, Biber J, Sorribas V, and Murer H
- Subjects
- Animals, Humans, Kidney metabolism, Kidney Diseases metabolism, Phosphate Transport Proteins metabolism, Phosphates metabolism
- Abstract
Over the past 25 years, successive cloning of SLC34A1, SLC34A2 and SLC34A3, which encode the sodium-dependent inorganic phosphate (P
i ) cotransport proteins 2a-2c, has facilitated the identification of molecular mechanisms that underlie the regulation of renal and intestinal Pi transport. Pi and various hormones, including parathyroid hormone and phosphatonins, such as fibroblast growth factor 23, regulate the activity of these Pi transporters through transcriptional, translational and post-translational mechanisms involving interactions with PDZ domain-containing proteins, lipid microdomains and acute trafficking of the transporters via endocytosis and exocytosis. In humans and rodents, mutations in any of the three transporters lead to dysregulation of epithelial Pi transport with effects on serum Pi levels and can cause cardiovascular and musculoskeletal damage, illustrating the importance of these transporters in the maintenance of local and systemic Pi homeostasis. Functional and structural studies have provided insights into the mechanism by which these proteins transport Pi , whereas in vivo and ex vivo cell culture studies have identified several small molecules that can modify their transport function. These small molecules represent potential new drugs to help maintain Pi homeostasis in patients with chronic kidney disease - a condition that is associated with hyperphosphataemia and severe cardiovascular and skeletal consequences.- Published
- 2019
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4. Intestinal Response to Acute Intragastric and Intravenous Administration of Phosphate in Rats.
- Author
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Layunta E, Pastor Arroyo EM, Kägi L, Thomas L, Levi M, Hernando N, and Wagner CA
- Subjects
- Administration, Intravenous, Animals, Glucose metabolism, Intestinal Mucosa cytology, Intestinal Mucosa metabolism, Male, RNA, Messenger metabolism, Rats, Rats, Wistar, Sodium-Phosphate Cotransporter Proteins, Type IIb metabolism, Transcription Factor Pit-1 genetics, Transcription Factor Pit-1 metabolism, Down-Regulation drug effects, Intestinal Mucosa drug effects, Phosphates pharmacology
- Abstract
Background/aims: Phosphate (Pi) homeostasis is controlled by the intestine and kidneys whose capacities to transport Pi are under endocrine control. Several studies point to intestinal absorption as a therapeutic target to modulate Pi homeostasis. The small intestine is responsible for almost all Pi absorption in the gut, a process involving Na
+ -dependent and independent mechanisms. Three Na+ -dependent Pi cotransporters have been described in the gastrointestinal tract: NaPi-IIb (a SLC34 member) and Pit-1 and Pit-2 (SLC20 transporters). We recently analysed the acute hormonal and renal response to intragastric (i.g) and intravenous (i.v) Pi-loading. This study demonstrated that the kidney quickly adapts to Pi-loading, with changes manifesting earlier in the i.v than i.g intervention. The aim of this work was to extend the previous studies in order to investigate the acute adaptation of intestinal transport of Pi and expression of intestinal Na+ /Pi-cotransporters in response to acute Pi-loading., Methods: Duodenal and jejunal mucosa was collected 40 minutes and/or 4 hours after administration (i.g and i.v) of either NaCl or Pi to anaesthetized rats. Uptakes of Pi and protein expression of Na+ /Pi cotransporters were measured in brush border membrane vesicles (BBMV); the cotransporters' mRNA abundance was quantified by real-time PCR in total RNA extracted from whole mucosa., Results: Pi-loading did not modify transport of Pi in duodenal and jejunal BBMV 4 hours after treatment. Administration of Pi did not alter either the intestinal expression of NaPi-IIb and Pit-2 mRNAs, whereas Pit-1 mRNA expression was only regulated (diminished) in duodenum collected 4 hours after i.g Pi-loading. NaPi-IIb protein expression was decreased in duodenum 4 hours upon i.v Pi infusion, whereas the duodenal and jejunal abundance of the cotransporter was unaffected by i.g administration of Pi., Conclusion: Together, these data suggest that the intestine responds acutely to Pi-loading, though this response seems slower than the renal adaptation., Competing Interests: The authors declare that they have no competing financial interests., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)- Published
- 2019
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5. Intestinal phosphate absorption is mediated by multiple transport systems in rats.
- Author
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Candeal E, Caldas YA, Guillén N, Levi M, and Sorribas V
- Subjects
- Animals, Biological Transport, Duodenum drug effects, Hydrogen-Ion Concentration, Intestinal Absorption drug effects, Jejunum drug effects, Microvilli drug effects, Phosphates administration & dosage, Rats, Sodium-Phosphate Cotransporter Proteins, Type IIb genetics, Sodium-Phosphate Cotransporter Proteins, Type IIb metabolism, Duodenum metabolism, Intestinal Absorption physiology, Jejunum metabolism, Microvilli metabolism, Phosphates metabolism
- Abstract
Apical inorganic phosphate (P
i ) transport in the small intestine seems to be mainly mediated by the sodium/Pi cotransporter NaPi2b. To verify this role, we have studied the combined effects of pH, phosphonoformate, and Pi deprivation on intestinal Pi transport. Rats were fed, ad libitum, three fodders containing 1.2, 0.6, or 0.1% Pi for 1, 5, or 10 days. Pi deprivation (0.1%) increased both sodium-activated and sodium-independent Pi transport in brush-border membrane vesicles from the duodenum and jejunum for all three times. Alkaline pH inhibited Pi transport, despite the increasing concentration of [Formula: see text] (NaPi2b substrate), whereas acidity increased transport when the concentration of the PiT1/PiT2 substrate, [Formula: see text], was at its highest. The effect of Pi deprivation was maximal at acid pH, but both basal and upregulated transport were inhibited (70%) with phosphonoformate, an inhibitor of NaPi2b. PiT2 and NaPi2b protein abundance increased after 24 h of Pi deprivation in the duodenum, jejunum, and ileum, whereas PiT1 required 5-10 days in the duodenum and jejunum. Therefore, whereas transporter expressions are partially correlated with Pi transport adaptation, the pH effect precludes NaPi2b, and phosphonoformic acid precludes PiT1 and PiT2 as the main transporters. Transport and transporter expression were also inconsistent when feeding was limited to 4 h daily, because the 1.2% Pi diet paradoxically increased Pi transport in the duodenum and jejunum, but NaPi2b and PiT1 expressions only increased with the 0.1% diet. These findings suggest the presence of a major transporter that carries [Formula: see text] and is inhibited by phosphonoformate. NEW & NOTEWORTHY The combined effects of dietary inorganic phosphate (Pi ) content, pH, and phosphonoformate inhibition suggest that the resulting apical Pi transport in the small intestine cannot be fully explained by the presence of NaPi2b, PiT1, or PiT2. We provide evidence of the presence of a new sodium-coupled Pi transporter that uses [Formula: see text] as the preferred substrate and is inhibited by phosphonoformate, and its expression correlates with Pi transport in all assayed conditions., (Copyright © 2017 the American Physiological Society.)- Published
- 2017
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6. Renal control of calcium, phosphate, and magnesium homeostasis.
- Author
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Blaine J, Chonchol M, and Levi M
- Subjects
- Animals, Calcium blood, Calcium urine, Gastrointestinal Absorption, Glomerular Filtration Rate, Homeostasis, Humans, Kidney physiopathology, Magnesium blood, Magnesium urine, Metabolic Diseases metabolism, Metabolic Diseases physiopathology, Phosphates blood, Phosphates urine, Renal Elimination, Renal Reabsorption, Calcium metabolism, Kidney metabolism, Magnesium metabolism, Phosphates metabolism
- Abstract
Calcium, phosphate, and magnesium are multivalent cations that are important for many biologic and cellular functions. The kidneys play a central role in the homeostasis of these ions. Gastrointestinal absorption is balanced by renal excretion. When body stores of these ions decline significantly, gastrointestinal absorption, bone resorption, and renal tubular reabsorption increase to normalize their levels. Renal regulation of these ions occurs through glomerular filtration and tubular reabsorption and/or secretion and is therefore an important determinant of plasma ion concentration. Under physiologic conditions, the whole body balance of calcium, phosphate, and magnesium is maintained by fine adjustments of urinary excretion to equal the net intake. This review discusses how calcium, phosphate, and magnesium are handled by the kidneys., (Copyright © 2015 by the American Society of Nephrology.)
- Published
- 2015
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7. Estrogen directly and specifically downregulates NaPi-IIa through the activation of both estrogen receptor isoforms (ERα and ERβ) in rat kidney proximal tubule.
- Author
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Burris D, Webster R, Sheriff S, Faroqui R, Levi M, Hawse JR, and Amlal H
- Subjects
- Animals, Cell Line, Chlorides urine, Eating, Estrogen Receptor alpha metabolism, Estrogen Receptor beta metabolism, Female, Humans, Kidney Cortex metabolism, Phosphate Transport Proteins metabolism, Random Allocation, Rats, Sprague-Dawley, Estrogens physiology, Kidney Tubules, Proximal metabolism, Phosphates metabolism, Sodium-Phosphate Cotransporter Proteins, Type IIa metabolism
- Abstract
We have previously demonstrated that estrogen (E2) downregulates phosphate transporter NaPi-IIa and causes phosphaturia and hypophosphatemia in ovariectomized rats. In the present study, we examined whether E2 directly targets NaPi-IIa in the proximal tubule (PT) and studied the respective roles of estrogen receptor isoforms (ERα and ERβ) in the downregulation of NaPi-IIa using both in vivo and an in vitro expression systems. We found that estrogen specifically downregulates NaPi-IIa but not NaPi-IIc or Pit2 in the kidney cortex. Proximal tubules incubated in a "shake" suspension with E2 for 24 h exhibited a dose-dependent decrease in NaPi-IIa protein abundance. Results from OVX rats treated with specific agonists for either ERα [4,4',4″;-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol, PPT] or ERβ [4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol, DPN] or both (PPT + DPN), indicated that only the latter caused a sharp downregulation of NaPi-IIa, along with significant phosphaturia and hypophosphatemia. Lastly, heterologous expression studies demonstrated that estrogen downregulated NaPi-IIa only in U20S cells expressing both ERα and ERβ, but not in cells expressing either receptor alone. In conclusion, these studies demonstrate that rat PT cells express both ERα and ERβ and that E2 induces phosphaturia by directly and specifically targeting NaPi-IIa in the PT cells. This effect is mediated via a mechanism involving coactivation of both ERα and ERβ, which likely form a functional heterodimer complex in the rat kidney proximal tubule., (Copyright © 2015 the American Physiological Society.)
- Published
- 2015
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8. Na+-independent phosphate transport in Caco2BBE cells.
- Author
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Candeal E, Caldas YA, Guillén N, Levi M, and Sorribas V
- Subjects
- Animals, Biological Transport, Caco-2 Cells, Humans, Hydrogen-Ion Concentration, Intestines drug effects, Kinetics, Male, Membrane Transport Modulators pharmacology, Phosphate Transport Proteins antagonists & inhibitors, Rats, Wistar, Intestinal Mucosa metabolism, Phosphate Transport Proteins metabolism, Phosphates metabolism
- Abstract
Pi transport in epithelia has both Na(+)-dependent and Na(+)-independent components, but so far only Na(+)-dependent transporters have been characterized in detail and molecularly identified. Consequently, in the present study, we initiated the characterization and analysis of intestinal Na(+)-independent Pi transport using an in vitro model, Caco2BBE cells. Only Na(+)-independent Pi uptake was observed in these cells, and Pi uptake was dramatically increased when cells were incubated in high-Pi DMEM (4 mM) from 1 day to several days. No response to low-Pi medium was observed. The increased Pi transport was mainly caused by Vmax changes, and it was prevented by actinomycin D and cycloheximide. Pi transport in cells grown in 1 mM Pi (basal DMEM) decreased at pH > 7.5, and it was inhibited with proton ionophores. Pi transport in cells incubated with 4 mM Pi increased with alkaline pH, suggesting a preference for divalent phosphate. Pi uptake in cells in 1 mM Pi was completely inhibited only by Pi and partially inhibited by phosphonoformate, oxalate, DIDS, SITS, SO4 (2-), HCO3 (-), and arsenate. This inhibition pattern suggests that more than one Pi transporter is active in cells maintained with 1 mM Pi. Phosphate transport from cells maintained at 4 mM Pi was only partially inhibited by phosphonoformate, oxalate, and arsenate. Attempts to identify the responsible transporters showed that multifunctional anion exchangers of the Slc26 family as well as members of Slc17, Slc20, and Slc37 and the Pi exporter xenotropic and polytropic retrovirus receptor 1 are not involved., (Copyright © 2014 the American Physiological Society.)
- Published
- 2014
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9. Renal phosphate wasting in the absence of adenylyl cyclase 6.
- Author
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Fenton RA, Murray F, Dominguez Rieg JA, Tang T, Levi M, and Rieg T
- Subjects
- Adenylyl Cyclases metabolism, Animals, Cyclic AMP metabolism, Female, Fibroblast Growth Factor-23, Fibroblast Growth Factors metabolism, Homeostasis, Hyperparathyroidism genetics, Hyperphosphatemia genetics, Immunohistochemistry, Lysosomes metabolism, Male, Mice, Mice, Transgenic, Parathyroid Hormone metabolism, Phenotype, Phosphates urine, Protein Transport, Sodium-Phosphate Cotransporter Proteins, Type IIa metabolism, Adenylyl Cyclases genetics, Kidney metabolism, Kidney Diseases genetics, Phosphates chemistry
- Abstract
Parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF-23) enhance phosphate excretion by the proximal tubule of the kidney by retrieval of the sodium-dependent phosphate transporters (Npt2a and Npt2c) from the apical plasma membrane. PTH activates adenylyl cyclase (AC) through PTH 1 receptors and stimulates the cAMP/PKA signaling pathway. However, the precise role and isoform(s) of AC in phosphate homeostasis are not known. We report here that mice lacking AC6 (AC6(-/-)) have increased plasma PTH and FGF-23 levels compared with wild-type (WT) mice but comparable plasma phosphate concentrations. Acute activation of the calcium-sensing receptor or feeding a zero phosphate diet almost completely suppressed plasma PTH levels in both AC6(-/-) and WT mice, indicating a secondary cause for hyperparathyroidism. Pharmacologic blockade of FGF receptors resulted in a comparable increase in plasma phosphate between genotypes, whereas urinary phosphate remained significantly higher in AC6(-/-) mice. Compared with WT mice, AC6(-/-) mice had reduced renal Npt2a and Npt2c protein abundance, with approximately 80% of Npt2a residing in lysosomes. WT mice responded to exogenous PTH with redistribution of Npt2a from proximal tubule microvilli to intracellular compartments and lysosomes alongside a PTH-induced dose-response relationship for fractional phosphate excretion and urinary cAMP excretion. These responses were absent in AC6(-/-) mice. In conclusion, AC6 in the proximal tubule modulates cAMP formation, Npt2a trafficking, and urinary phosphate excretion, which are highlighted by renal phosphate wasting in AC6(-/-) mice., (Copyright © 2014 by the American Society of Nephrology.)
- Published
- 2014
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10. Inorganic phosphate modulates the expression of the NaPi-2a transporter in the trans-Golgi network and the interaction with PIST in the proximal tubule.
- Author
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Lanaspa MA, Caldas YA, Breusegem SY, Andrés-Hernando A, Cicerchi C, Levi M, and Sorribas V
- Subjects
- Animals, Biological Transport, Cell Membrane metabolism, Culture Media pharmacology, Endocytosis, Endosomes metabolism, Lysosomes metabolism, Male, Opossums, Rats, Rats, Wistar, Kidney Tubules, Proximal metabolism, Phosphates metabolism, Sodium-Phosphate Cotransporter Proteins, Type IIa metabolism, trans-Golgi Network metabolism
- Abstract
Inorganic phosphate (Pi) homeostasis is maintained by the tight regulation of renal Pi excretion versus reabsorption rates that are in turn modulated by adjusting the number of Pi transporters (mainly NaPi-2a) in the proximal tubules. In response to some hormones and a high dietary Pi content, NaPi-2a is endocytosed and degraded in the lysosomes; however, we show here that some NaPi-2a molecules are targeted to the trans-Golgi network (TGN) during the endocytosis. In the TGN, NaPi-2a interacts with PIST (PDZ-domain protein interacting specifically with TC10), a TGN-resident PDZ-domain-containing protein. The extension of the interaction is proportional to the expression of NaPi-2a in the TGN, and, consistent with that, it is increased with a high Pi diet. When overexpressed in opossum kidney (OK) cells, PIST retains NaPi-2a in the TGN and inhibits Na-dependent Pi transport. Overexpression of PIST also prevents the adaptation of OK cells to a low Pi culture medium. Our data supports the view that NaPi-2a is subjected to retrograde trafficking from the plasma membrane to the TGN using one of the machineries involved in endosomal transport and explains the reported expression of NaPi-2a in the TGN.
- Published
- 2013
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11. The flux of phosphate: rapid evolution.
- Author
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Lederer E and Levi M
- Subjects
- Cardiovascular Diseases metabolism, Chronic Kidney Disease-Mineral and Bone Disorder metabolism, Humans, Renal Insufficiency, Chronic physiopathology, Risk Factors, Syndrome, Bone Diseases, Metabolic metabolism, Homeostasis, Phosphates metabolism, Renal Insufficiency, Chronic metabolism
- Published
- 2011
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12. Intestinal phosphate transport.
- Author
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Sabbagh Y, Giral H, Caldas Y, Levi M, and Schiavi SC
- Subjects
- Absorption genetics, Absorption physiology, Animals, Biological Transport, Active genetics, Biological Transport, Active physiology, Gene Expression Regulation, Humans, Intestinal Absorption genetics, Intestine, Small metabolism, Ion Transport genetics, Ion Transport physiology, Kidney metabolism, Renal Insufficiency, Chronic metabolism, Sodium-Phosphate Cotransporter Proteins genetics, Sodium-Phosphate Cotransporter Proteins, Type III genetics, Sodium-Phosphate Cotransporter Proteins, Type III metabolism, Sodium-Phosphate Cotransporter Proteins, Type IIb genetics, Sodium-Phosphate Cotransporter Proteins, Type IIb metabolism, Intestinal Absorption physiology, Phosphates metabolism, Sodium-Phosphate Cotransporter Proteins metabolism
- Abstract
Phosphate is absorbed in the small intestine by a minimum of 2 distinct mechanisms: paracellular phosphate transport which is dependent on passive diffusion, and active transport which occurs through the sodium-dependent phosphate cotransporters. Despite evidence emerging for other ions, regulation of the phosphate-specific paracellular pathways remains largely unexplored. In contrast, there is a growing body of evidence that active transport through the sodium-dependent phosphate cotransporter, Npt2b, is highly regulated by a diverse set of hormones and dietary conditions. Furthermore, conditional knockout of Npt2b suggests that it plays an important role in maintenance of phosphate homeostasis by coordinating intestinal phosphate absorption with renal phosphate reabsorption. The knockout mouse also suggests that Npt2b is responsible for the majority of sodium-dependent phosphate uptake. The type-III sodium-dependent phosphate transporters, Pit1 and Pit2, contribute to a minor role in total phosphate uptake. Despite coexpression along the apical membrane, differential responses of Pit1 and Npt2b regulation to chronic versus dietary changes illustrates another layer of phosphate transport control. Finally, a major problem in patients with CKD is management of hyperphosphatemia. The present evidence suggests that targeting key regulatory pathways of intestinal phosphate transport may provide novel therapeutic approaches for patients with CKD., (Copyright © 2011 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)
- Published
- 2011
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13. Regulation of rat intestinal Na-dependent phosphate transporters by dietary phosphate.
- Author
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Giral H, Caldas Y, Sutherland E, Wilson P, Breusegem S, Barry N, Blaine J, Jiang T, Wang XX, and Levi M
- Subjects
- Animals, Blotting, Western, Cell Membrane metabolism, Duodenum drug effects, Duodenum metabolism, Enterocytes metabolism, Jejunum drug effects, Jejunum metabolism, Male, Microscopy, Fluorescence, Microvilli drug effects, Microvilli metabolism, RNA, Messenger biosynthesis, RNA, Messenger genetics, Rats, Rats, Sprague-Dawley, Sodium-Phosphate Cotransporter Proteins, Type III biosynthesis, Sodium-Phosphate Cotransporter Proteins, Type III genetics, Sodium-Phosphate Cotransporter Proteins, Type IIb biosynthesis, Intestine, Small drug effects, Intestine, Small metabolism, Phosphates pharmacology, Phosphorus, Dietary pharmacology, Sodium-Phosphate Cotransporter Proteins biosynthesis
- Abstract
Hyperphosphatemia associated with chronic kidney disease is one of the factors that can promote vascular calcification, and intestinal P(i) absorption is one of the pharmacological targets that prevents it. The type II Na-P(i) cotransporter NaPi-2b is the major transporter that mediates P(i) reabsorption in the intestine. The potential role and regulation of other Na-P(i) transporters remain unknown. We have identified expression of the type III Na-P(i) cotransporter PiT-1 in the apical membrane of enterocytes. Na-P(i) transport activity and NaPi-2b and PiT-1 proteins are mostly expressed in the duodenum and jejunum of rat small intestine; their expression is negligible in the ileum. In response to a chronic low-P(i) diet, there is an adaptive response restricted to the jejunum, with increased brush border membrane (BBM) Na-P(i) transport activity and NaPi-2b, but not PiT-1, protein and mRNA abundance. However, in rats acutely switched from a low- to a high-P(i) diet, there is an increase in BBM Na-P(i) transport activity in the duodenum that is associated with an increase in BBM NaPi-2b protein abundance. Acute adaptive upregulation is restricted to the duodenum and induces an increase in serum P(i) that produces a transient postprandial hyperphosphatemia. Our study, therefore, indicates that Na-P(i) transport activity and NaPi-2b protein expression are differentially regulated in the duodenum vs. the jejunum and that postprandial upregulation of NaPi-2b could be a potential target for treatment of hyperphosphatemia.
- Published
- 2009
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14. Vascular smooth muscle cell calcification and SLC20 inorganic phosphate transporters: effects of PDGF, TNF-alpha, and Pi.
- Author
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Villa-Bellosta R, Levi M, and Sorribas V
- Subjects
- Animals, Becaplermin, Calcinosis etiology, Muscle, Smooth, Vascular drug effects, Muscle, Smooth, Vascular pathology, Phosphates metabolism, Proto-Oncogene Proteins c-sis, Rats, Muscle, Smooth, Vascular metabolism, Phosphates pharmacology, Platelet-Derived Growth Factor pharmacology, Sodium-Phosphate Cotransporter Proteins, Type III physiology, Tumor Necrosis Factor-alpha pharmacology
- Abstract
Pi transport by vascular smooth muscle cells (VSMC) has been proposed to play an important role in the pathogenesis of vascular calcification. In this study, we have determined the correlation between calcification induced by Pi, platelet-derived growth factor (PDGF)-BB, and tumor necrosis factor-alpha and Pi transport activity in primary cultures of rat aortic VSMC. These agents induced calcification and increased the expression of Cbfa1, Msx2, and Bmp2 osteogene messenger RNA in rat aortic VSMC, while Pi transport rate was not modified per milligram of protein. Only PDGF increased Pi transport when it was expressed per unit of DNA, as PDGF also increased total cell protein by 100%, while DNA content and number of cells were not modified. PDGF increased the expression of the Pi transporter, Pit-1, but membrane protein biotinylation showed that Pit-1 abundance was not modified in the cell surface. Immunofluorescence revealed that, under basal conditions, Pit-1 is only slightly expressed at the cell membrane, but strongly expressed inside the cell. The intracellular signal colocalizes with endoplasmic reticulum (ER) markers, and PDGF increases Pit-1 expression in the ER but not the cell membrane. In conclusion, Pi transport across the plasma membrane does not correlate directly with calcification, but the expression of Pit-1 in the ER opens new possibilities for the study of the pathogenesis of vascular calcification.
- Published
- 2009
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15. The Na+-Pi cotransporter PiT-2 (SLC20A2) is expressed in the apical membrane of rat renal proximal tubules and regulated by dietary Pi.
- Author
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Villa-Bellosta R, Ravera S, Sorribas V, Stange G, Levi M, Murer H, Biber J, and Forster IC
- Subjects
- Adaptation, Physiological, Animals, Biological Transport, Blotting, Western, Cell Polarity, Immunohistochemistry, Male, Microvilli metabolism, Phosphates administration & dosage, Phosphates deficiency, Rats, Rats, Wistar, Sodium-Phosphate Cotransporter Proteins, Type II metabolism, Time Factors, Cell Membrane metabolism, Diet, Kidney Tubules, Proximal metabolism, Phosphates metabolism, Sodium-Phosphate Cotransporter Proteins, Type III metabolism
- Abstract
The principal mediators of renal phosphate (P(i)) reabsorption are the SLC34 family proteins NaPi-IIa and NaPi-IIc, localized to the proximal tubule (PT) apical membrane. Their abundance is regulated by circulatory factors and dietary P(i). Although their physiological importance has been confirmed in knockout animal studies, significant P(i) reabsorptive capacity remains, which suggests the involvement of other secondary-active P(i) transporters along the nephron. Here we show that a member of the SLC20 gene family (PiT-2) is localized to the brush-border membrane (BBM) of the PT epithelia and that its abundance, confirmed by Western blot and immunohistochemistry of rat kidney slices, is regulated by dietary P(i). In rats treated chronically on a high-P(i) (1.2%) diet, there was a marked decrease in the apparent abundance of PiT-2 protein in kidney slices compared with those from rats kept on a chronic low-P(i) (0.1%) diet. In Western blots of BBM from rats that were switched from a chronic low- to high-P(i) diet, NaPi-IIa showed rapid downregulation after 2 h; PiT-2 was also significantly downregulated at 24 h and NaPi-IIc after 48 h. For the converse dietary regime, NaPi-IIa showed adaptation within 8 h, whereas PiT-2 and NaPi-IIc showed a slower adaptive trend. Our findings suggest that PiT-2, until now considered as a ubiquitously expressed P(i) housekeeping transporter, is a novel mediator of P(i) reabsorption in the PT under conditions of acute P(i) deprivation, but with a different adaptive time course from NaPi-IIa and NaPi-IIc.
- Published
- 2009
- Full Text
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16. Renal phosphate-transporter regulatory proteins and nephrolithiasis.
- Author
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Levi M and Breusegem S
- Subjects
- Animals, Biological Transport genetics, Cyclic AMP metabolism, Humans, Hypercalciuria genetics, Kidney metabolism, Mice, Mice, Knockout, Mutation, Phosphoproteins deficiency, Bone Demineralization, Pathologic genetics, Nephrolithiasis genetics, Phosphates metabolism, Phosphoproteins genetics, Sodium-Hydrogen Exchangers genetics
- Published
- 2008
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17. Insulin attenuates vascular smooth muscle calcification but increases vascular smooth muscle cell phosphate transport.
- Author
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Wang CC, Sorribas V, Sharma G, Levi M, and Draznin B
- Subjects
- Animals, Cells, Cultured, Enzyme Inhibitors pharmacology, Insulin Resistance, Kinetics, Rats, Risk, Signal Transduction, Sodium-Phosphate Cotransporter Proteins chemistry, Transcription Factor Pit-1 metabolism, Calcium metabolism, Insulin metabolism, Muscle, Smooth, Vascular metabolism, Phosphates metabolism
- Abstract
Medial artery vascular smooth muscle cell (VSMC) calcification increases the risk of cardiovascular mortality in type 2 diabetes. However, the influence of insulin on VSMC calcification is unclear. We explored the effects of insulin on rat VSMC calcification in vitro and found that in a dose-dependent fashion, insulin attenuates VSMC calcification induced by high phosphate conditions as quantified by the o-cresolphthalein calcium (OCPC) method. In an in vitro model of insulin resistance in which cells are exposed to elevated insulin concentrations and the PI 3-kinase pathway is selectively inhibited, increased VSMC calcification was observed, suggesting that the PI 3-kinase pathway is involved in this attenuating effect of insulin. We postulated that insulin may also have an effect on phosphate or calcium transport in VSMC. We found that insulin increases phosphate transport at 3 and 24 h. This effect was mediated by increased Vmax for phosphate transport but not Km. Because type III sodium-phosphate co-transporters Pit-1 and Pit-2 are found in VSMC, we examined their expression by Western blot and real-time RT-PCR. Insulin stimulates Pit-1 mRNA modestly (*p<0.01 versus control), an effect inhibited by PD98059 but not by wortmannin. Pit-1 protein expression is induced by insulin, an effect also inhibited by PD98059 (*p<0.001 versus insulin alone). Our results suggest a role for insulin in attenuating VSMC calcification which may be disrupted in selective insulin signaling impairment seen in insulin resistance. This effect of insulin contrasts with its effect to induce phosphate transport in VSMC.
- Published
- 2007
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18. Renal phosphate-wasting disorders.
- Author
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Levi M, Blaine J, Breusegem S, Takahashi H, Sorribas V, and Barry N
- Subjects
- Animals, Humans, Hypophosphatemia, Familial genetics, Ion Transport genetics, Mutation, Hypophosphatemia, Familial metabolism, Kidney metabolism, Phosphates metabolism
- Abstract
The renal regulation of phosphate is a complex process. Clinical disorders of phosphate handling have led to the identification of several genes and proteins involved in the maintenance of phosphate homeostasis. Further work is required to elucidate the precise pathways that regulate renal phosphate transport.
- Published
- 2006
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19. Effect of ischemia reperfusion on sodium-dependent phosphate transport in renal brush border membranes.
- Author
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Khundmiri SJ, Asghar M, Banday AA, Khan F, Salim S, Levi M, and Yusufi AN
- Subjects
- Animals, Biological Transport, Blotting, Western, Ischemia pathology, Kidney Cortex metabolism, Kinetics, Microvilli, Phosphorus metabolism, Proline chemistry, Rats, Rats, Wistar, Renal Insufficiency metabolism, Reperfusion, Sodium chemistry, Thyroid Hormones metabolism, Time Factors, Triiodothyronine metabolism, Cell Membrane metabolism, Kidney metabolism, Phosphates chemistry, Reperfusion Injury
- Abstract
The effect of ischemia induced acute renal failure (ARF) on the transport of phosphate (Pi) after early (15-30 min) and prolonged (60 min) ischemia in the brush border membrane vesicles (BBMV) from rat renal cortex was studied. Sodium-dependent transport of Pi declined significantly and progressively due to ischemia. Western blot analysis of BBM from ischemic rats showed decreased expression of NaPi-2. A compensatory increase was observed in Pi uptake in BBMV from contralateral kidneys. There was no significant difference in NaPi-2 expression between BBMV from sham and contralateral kidneys. Early blood reperfusion for 15 min after 30 min ischemia caused further decline in Pi uptake. Prolonged reperfusion for 120 min caused partial reversal of transport activities in 30-min ischemic rats. However, no improvement in the transport of Pi was observed in 60-min ischemic rats after 120 min of blood reperfusion. Kinetic studies showed that the effect of ischemia and blood reperfusion was dependent on the Vmax of the Na-Pi transporter. Western blot analysis showed increased expression of NaPi-2 in the BBMs from ischemia-reperfusion animals. Further, a shift in the association of Na ions to transport one molecule of Pi was observed under different extracellular Na concentrations [Na]o. Feeding rats with low Pi diet and/or treatment with thyroid hormone (T3) prior to ischemia resulted in increased basal Pi transport. Ischemia caused similar decline in Pi transport in BBM from LPD and/or T3 animals. However, recovery in these animals was faster than the normal Pi diet fed (NPD) animals. The study suggests a change in the intrinsic properties of the Na-Pi transporter in rat kidneys due to ischemia. The study also indicates that treatment with T3 and feeding LPD prior to ischemia caused faster recovery of phosphate uptake due to ischemia-reperfusion injury.
- Published
- 2005
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20. Regulation of the renal sodium-dependent phosphate cotransporter NaPi2 (Npt2) in acute renal failure due to ischemia and reperfusion.
- Author
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Rubinger D, Wald H, Gimelreich D, Halaihel N, Rogers T, Levi M, and Popovtzer MM
- Subjects
- Acute Kidney Injury etiology, Animals, Gene Expression Regulation, Male, Parathyroidectomy, RNA, Messenger metabolism, Rats, Rats, Wistar, Reperfusion Injury complications, Sodium-Phosphate Cotransporter Proteins, Type IIa deficiency, Sodium-Phosphate Cotransporter Proteins, Type IIa genetics, Acute Kidney Injury metabolism, Phosphates metabolism, Reperfusion Injury metabolism, Sodium-Phosphate Cotransporter Proteins, Type IIa metabolism
- Abstract
Background: Acute renal failure (ARF) is associated with hyperphosphatemia and decreased urinary phosphate excretion. The present study was undertaken to characterize the effects of ARF due to ischemia and reperfusion on renal phosphate transport and on gene and protein expression of type IIa NaPi cotransporter (Npt2) the physiologically most relevant renal sodium-dependent phosphate cotransporter., Methods: The following groups of rats with intact parathyroid glands were studied: (1) sham operated (sham); (2) after 1 h ischemia by bilateral renal artery clamping (I), and after 1 h ischemia and reperfusion of 1 h (I + R 1 h); (3) 24 h (I + R 24 h); (4) 48 h (I + R 48 h), and (5) 72 h (I + R 72 h) duration. The effect of ARF on Npt2 mRNA and protein expression was also examined after parathyroidectomy (PTX) of 2 and 4 days' duration., Results: Ischemia and reperfusion were associated with increases in plasma creatinine, hyperphosphatemia, and with decreased tubular phosphate reabsorption. Npt2 mRNA was significantly downregulated in the cortex, maximal at 24 and 48 h of reperfusion. The degree of Npt2 mRNA downregulation was not affected by PTX of 2-4 days' duration. The abundance of Npt2 protein in proximal tubular apical brush border membrane was markedly decreased after reperfusion. Npt2 protein, however, was more abundant in PTX animals than in those with intact parathyroids and a similar degree of renal insufficiency. The immunohistochemical analysis of proximal tubular apical brush border membrane showed a progressive decrease of Npt2 protein labeling after ischemia and reperfusion, with progressive regeneration after 72 h., Conclusion: These results suggest that downregulation of Npt2 protein may contribute to the decreased tubular reabsorption of phosphate in acute ischemic renal failure and hyperphosphatemia.
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- 2005
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21. Recovery of renal tubule phosphate reabsorption despite reduced levels of sodium-phosphate transporter.
- Author
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Friedlaender MM, Wald H, Dranitzky-Elhalel M, Levi M, and Popovtzer MM
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- Absorption, Animals, Calcium blood, Creatinine metabolism, Cyclic AMP urine, Electrophoresis, Polyacrylamide Gel, Immunoblotting, Male, Microvilli drug effects, Parathyroid Hormone pharmacology, Parathyroidectomy, Phosphates blood, Rats, Sodium-Phosphate Cotransporter Proteins, Sodium-Phosphate Cotransporter Proteins, Type IIa, Kidney Tubules metabolism, Phosphates metabolism, Symporters metabolism
- Abstract
Background: The acute effect of parathyroid hormone (PTH) on phosphate transport has been reported to be mediated by rapid downregulation of sodium-phosphate transporter (NaPi-IIa) protein, but the association was observed with pharmacological doses of PTH., Objective: To explore the effects of physiological doses of PTH on NaPi-IIa protein and its relationship to phosphate transport., Methods: Acute clearance studies were performed in parathyroidectomized rats given a bolus i.v. physiological dose (1 microg) of bovine PTH(1-34) and NaPi-IIa protein concentrations were examined at different time intervals., Results: Fractional excretion of phosphate increased from 0.031+/-0.006 (mean+/-S.E.) to 0.238+/-0.059 (P<0.01 compared with baseline and compared with controls) at 40 min and returned to control values by 120 min. Urinary cAMP concentrations were increased at 20 min only. Superficial cortex brush-border membrane (BBM) NaPi-IIa protein was decreased from baseline at both 40 and 120 min (P<0.01) and did not recover at 240 min (P<0.01 compared with baseline and compared with controls)., Conclusion: These results confirm that PTH, even in physiological dosage, causes a rapid decrease in BBM NaPi-IIa, but subsequent recovery of phosphate reabsorption is poorly correlated with BBM concentrations of NaPi-IIa protein. This suggests that transport mechanisms other than NaPi-IIa are important in renal phosphate reabsorption.
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- 2004
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22. Regulation of NaPi-IIa mRNA and transporter protein in chronic renal failure: role of parathyroid hormone (PTH) and dietary phosphate (Pi).
- Author
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Elhalel MD, Wald H, Rubinger D, Gal-Moscovici A, Inoue M, Levi M, and Popovtzer MM
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- Administration, Oral, Animals, Male, Metabolic Clearance Rate, RNA, Messenger metabolism, Rats, Sodium-Phosphate Cotransporter Proteins, Sodium-Phosphate Cotransporter Proteins, Type IIa, Kidney Failure, Chronic metabolism, Parathyroid Hormone metabolism, Parathyroid Hormone-Related Protein metabolism, Phosphates administration & dosage, Phosphates deficiency, Symporters metabolism
- Abstract
Chronic renal failure (CRF) is associated with a high fractional phosphate excretion (FEPi), secondary hyperparathyroidism, and resistance to parathyroid hormone (PTH). This study was undertaken to characterize the role of PTH and dietary Pi in the regulation of PTH/PTH-related peptide receptor (PTHrP-R) mRNA and NaPi-IIa mRNA and protein in CRF. The following groups of rats were studied: (1) sham-operated (control); (2) CRF: 6 weeks after 5/6 nephrectomy (NPX); (3) NPX and parathyroidectomy (NPX + PTX); (4) NPX rats fed a low-Pi diet (NPX + LP); (5) sham-operated rats fed a low-Pi diet (control + LP); (6) sham-operated after PTX (control + PTX). Expression of NaPi-IIa mRNA and PTH/PTHrP-R mRNA was determined in the renal cortex by Northern hybridization. NaPi-IIa protein abundance was determined in cortical brush border membranes by immunoblotting. In NPX rats creatinine clearance decreased to 40 +/- 4%, PTH/PTHrP-R mRNA to 52.1 +/- 2% and NaPi-IIa mRNA to 41.2 +/- 5.5% of control. The PTH/PTHrP-R and NaPi-IIa mRNA in the NPX + PTX and NPX + LP group was similar to that in NPX. NaPi-IIa protein abundance was reduced in NPX compared with control, but was increased dramatically in NPX + PTX and NPX + LP compared to NPX, paralleled by a decrease in FEPi. These findings suggest that the elevated FEPi in CRF is maintained by decreased NaPi-IIa mRNA and NaPi-IIa protein abundance. In contrast, the observed decrease in FEPi with PTX or LP diet in CRF is mediated, at least partly, by increased NaPi-IIa protein abundance with no change in NaPi-IIa mRNA, suggesting post-transcriptional regulation of the NaPi-IIa transporter.
- Published
- 2004
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23. Partitioning of NaPi cotransporter in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched membrane domains modulates NaPi protein diffusion, clustering, and activity.
- Author
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Inoue M, Digman MA, Cheng M, Breusegem SY, Halaihel N, Sorribas V, Mantulin WW, Gratton E, Barry NP, and Levi M
- Subjects
- Animals, Biological Transport, Blotting, Western, Centrifugation, Density Gradient, Detergents pharmacology, Diffusion, Lipids chemistry, Male, Membrane Microdomains metabolism, Microscopy, Microvilli metabolism, Normal Distribution, Photons, Potassium chemistry, Potassium Deficiency metabolism, Protein Binding, Protein Processing, Post-Translational, Protein Structure, Tertiary, Rats, Rats, Sprague-Dawley, Sodium-Phosphate Cotransporter Proteins, Sodium-Phosphate Cotransporter Proteins, Type IIa, Ultracentrifugation, Cholesterol chemistry, Glycosphingolipids chemistry, Phosphates chemistry, Sphingomyelins chemistry, Symporters chemistry
- Abstract
In dietary potassium deficiency there is a decrease in the transport activity of the type IIa sodium/phosphate cotransporter protein (NaPi) despite an increase in its apical membrane abundance. This novel posttranslational regulation of NaPi activity is mediated by the increased glycosphingolipid content of the potassium-deficient apical membrane. However, the mechanisms by which these lipids modulate NaPi activity have not been determined. We determined if in potassium deficiency NaPi is increasingly partitioned in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched microdomains of the apical membrane and if the increased presence of NaPi in these microdomains modulates its activity. By using a detergent-free density gradient flotation technique, we found that 80% of the apical membrane NaPi partitions into the low density cholesterol-, sphingomyelin-, and GM1-enriched fractions characterized as "lipid raft" fractions. In potassium deficiency, a higher proportion of NaPi was localized in the lipid raft fractions. By combining fluorescence correlation spectroscopy and photon counting histogram methods for control and potassium-deficient apical membranes reconstituted into giant unilamellar vesicles, we showed a 2-fold decrease in lateral diffusion of NaPi protein and a greater than 2-fold increase in size of protein aggregates/clusters in potassium deficiency. Our results indicate that NaPi protein is localized in membrane microdomains, that in potassium deficiency a larger proportion of NaPi protein is present in these microdomains, and that NaPi lateral diffusion is slowed down and NaPi aggregation/clustering is increased in potassium deficiency, both of which could be associated with the decreased Na/Pi cotransport activity in potassium deficiency.
- Published
- 2004
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24. Regulation of renal NaPi-2 expression and tubular phosphate reabsorption by growth hormone in the juvenile rat.
- Author
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Woda CB, Halaihel N, Wilson PV, Haramati A, Levi M, and Mulroney SE
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- Animals, Male, Microvilli physiology, Parathyroid Hormone pharmacology, Rats, Rats, Wistar, Sodium-Phosphate Cotransporter Proteins, Sodium-Phosphate Cotransporter Proteins, Type IIa, Symporters pharmacology, Up-Regulation, Growth Hormone biosynthesis, Growth Hormone pharmacology, Kidney Tubules, Proximal physiology, Phosphates pharmacokinetics, Symporters biosynthesis
- Abstract
Growth hormone (GH) is an important factor in the developmental adaptation to enhance P(i) reabsorption; however, the nephron sites and mechanisms by which GH regulates renal P(i) uptake remain unclear and are the focus of the present study. Micropuncture experiments were performed after acute thyroparathyroidectomy in the presence and absence of parathyroid hormone (PTH) in adult (14- to 17-wk old), juvenile (4-wk old), and GH-suppressed juvenile male rats. While the phosphaturic effect of PTH was blunted in the juvenile rat compared with the adult, suppression of GH in the juvenile restored fractional P(i) excretion to adult levels. In the presence or absence of PTH, GH suppression in the juvenile rat caused a significant increase in the fractional P(i) delivery to the late proximal convoluted (PCT) and early distal tubule, so that delivery was not different from that in adults. These data were confirmed by P(i) uptake studies into brush-border membrane (BBM) vesicles. Immunofluorescence studies indicate increased BBM type IIa NaP(i) cotransporter (NaPi-2) expression in the juvenile compared with adult rat, and GH suppression reduced NaPi-2 expression to levels observed in the adult. GH replacement in the [N-acetyl-Tyr(1)-d-Arg(2)]-GRF-(1-29)-NH(2)-treated juveniles restored high NaPi-2 expression and P(i) uptake. Together, these novel results demonstrate that the presence of GH in the juvenile animal is crucial for the early developmental upregulation of BBM NaPi-2 and, most importantly, describe the enhanced P(i) reabsorption along the PCT and proximal straight nephron segments in the juvenile rat.
- Published
- 2004
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25. Central control of renal sodium-phosphate (NaPi-2) transporters.
- Author
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Mulroney SE, Woda CB, Halaihel N, Louie B, McDonnell K, Schulkin J, Haramati A, and Levi M
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- Adaptation, Physiological physiology, Animals, Homeostasis drug effects, Homeostasis physiology, Male, Phosphorus, Dietary metabolism, Phosphorus, Dietary pharmacology, Rats, Rats, Wistar, Sodium-Phosphate Cotransporter Proteins, Sodium-Phosphate Cotransporter Proteins, Type II, Brain metabolism, Kidney metabolism, Phosphates cerebrospinal fluid, Sodium metabolism, Symporters metabolism
- Abstract
Regulation of phosphate (Pi) reabsorption occurs through the up- and downregulation of the renal type-II sodium Pi cotransporters (NaPi-2). Recently, renal NaPi2-type expression has been identified in areas of the brain. The present study determined whether brain NaPi-2 is regulated by dietary Pi and whether the behavioral and renal adaptations to low-dietary Pi are controlled centrally. NaPi-2-like expression in the third ventricle (3V) and amygdala of juvenile Wistar rats was regulated by dietary Pi, as in the kidneys. When cerebrospinal fluid (CSF) Pi concentration was elevated by 3V injections of Pi in rats fed low-Pi diet (LPD), the behavioral and renal adaptations to LPD were abolished. Most importantly, NaPi-2 expression was markedly reduced not only in the brain, but also renal proximal tubules, despite the low plasma Pi milieu. This was confirmed by the significant reduction in the transport maximum for Pi (from 8.1+/-0.2 in LPD + veh 3V to 1.7+/-0.1 micromol Pi/ml glomerular filtration rate in LPD + 3V Pi, P < 0.001). These findings indicate that NaPi-2-like transporters in the brain are regulated by both dietary Pi and CSF Pi concentrations, and most significantly, that the central Pi milieu can regulate renal NaPi-2 expression. We hypothesize that central 3V NaPi-2 transporters may act as Pi sensors and help regulate both brain and whole body Pi homeostasis.
- Published
- 2004
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26. Visualizing the regulation of SLC34 proteins at the apical membrane
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Levi, Moshe and Gratton, Enrico
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,Nutrition ,Kidney Disease ,1.1 Normal biological development and functioning ,Underpinning research ,Animals ,Cell Membrane ,Fibroblast Growth Factor-23 ,Humans ,Kidney Tubules ,Proximal ,Phosphates ,Protein Transport ,Sodium-Phosphate Cotransporter Proteins ,Type II ,NaPi-PDZ protein interactions ,NaPi transporter diffusion and clustering ,NaPi-lipid interactions ,Fluorescence lifetime imaging microscopy ,Forster resonance energy transfer ,Fluctuation correlation spectroscopy ,Förster resonance energy transfer ,Physiology ,Human Movement and Sports Sciences ,Medical Physiology ,Biochemistry and cell biology ,Zoology ,Medical physiology - Abstract
The cloning of the renal NaPi-2a (SLC34A1) and NaPi-2c (SLC34A3) phosphate transporters has made it possible to characterize the molecular and biophysical regulation of renal proximal tubular reabsorption of inorganic phosphate (Pi). Dietary factors, such as Pi and K, and several hormones and phosphatonins, including parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and glucocorticoids, regulate the transporters through various transcriptional, translational, and post-translational mechanisms that involve acute trafficking via endocytosis or exocytosis, interactions with PDZ domain proteins, lipid microdomains, and diffusion and clustering in the apical brush border membrane. The visualization of these trafficking events by means of novel microscopy techniques that includes fluorescence lifetime imaging microscopy (FLIM), Förster resonance energy transfer (FRET), fluctuation correlation spectroscopy (FCS), and modulation tracking (MT), is the primary focus of this review.
- Published
- 2019
27. Constitutive depletion of Slc34a2/NaPi-IIb in rats causes perinatal mortality.
- Author
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Pastor-Arroyo, Eva Maria, Rodriguez, Josep M. Monné, Pellegrini, Giovanni, Bettoni, Carla, Levi, Moshe, Hernando, Nati, and Wagner, Carsten A.
- Subjects
PERINATAL death ,PHOSPHATES ,HYPERPHOSPHATEMIA ,CARDIOVASCULAR diseases risk factors ,LABORATORY rats - Abstract
Absorption of dietary phosphate (Pi) across intestinal epithelia is a regulated process mediated by transcellular and paracellular pathways. Although hyperphosphatemia is a risk factor for the development of cardiovascular disease, the amount of ingested Pi in a typical Western diet is above physiological needs. While blocking intestinal absorption has been suggested as a therapeutic approach to prevent hyperphosphatemia, a complete picture regarding the identity and regulation of the mechanism(s) responsible for intestinal absorption of Pi is missing. The Na
+ /Pi cotransporter NaPi-IIb is a secondary active transporter encoded by the Slc34a2 gene. This transporter has a wide tissue distribution and within the intestinal tract is located at the apical membrane of epithelial cells. Based on mouse models deficient in NaPi-IIb, this cotransporter is assumed to mediate the bulk of active intestinal absorption of Pi. However, whether or not this is also applicable to humans is unknown, since human patients with inactivating mutations in SLC34A2 have not been reported to suffer from Pi depletion. Thus, mice may not be the most appropriate experimental model for the translation of intestinal Pi handling to humans. Here, we describe the generation of a rat model with Crispr/Cas-driven constitutive depletion of Slc34a2. Slc34a2 heterozygous rats were indistinguishable from wild type animals under standard dietary conditions as well as upon 3 days feeding on low Pi. However, unlike in humans, homozygosity resulted in perinatal lethality. [ABSTRACT FROM AUTHOR]- Published
- 2021
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28. Identification and expression analysis of type II and type III Pi transporters in the opossum kidney cell line.
- Author
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Guillén, Natalia, Caldas, Yupanqui A., Levi, Moshe, and Sorribas, Víctor
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PHOSPHATES ,OPOSSUMS ,KIDNEY cell culture ,CELL lines ,RNA - Abstract
New Findings: What is the central question of this study?The opossum kidney (OK) cell line is the main in vitro model of proximal tubular Pi transport, but it is incomplete because only the NaPiIIa Pi transporter has been identified.What is the main finding and its importance?We have cloned and characterized the Pi transporters NaPiIIc, PiT1 and PiT2 from OK cells and have analysed the relevance of the four transporters to Pi transport. All four transporters are involved in the upregulated Pi transport of cells incubated using a low‐Pi medium, and only PiT1 is not involved in basal transport. The apical membrane of renal proximal tubular epithelial cells is the main controller of phosphate homeostasis, because it determines the rate of urinary Pi excretion. The opossum kidney (OK) cell line is a good model for studying this function, but only NaPiIIa (NaPi4) has been identified to date as a Pi transporter in this cell line. In this work, we have identified three additional Pi transporters that are present in OK cells: NaPiIIc, PiT1 and PiT2. All three sequences are similar to the corresponding orthologues, but PiT1 is missing the first transmembrane domain. Confluent cells exhibit characteristics of type II Pi transport, which increases with alkalinity and is inhibited by phosphonoformic acid (PFA), and they mainly express NaPiIIa and NaPiIIc, with a low abundance of PiT1 and PiT2. Proliferating cells show a higher expression of PiT1 and PiT2 and a low expression of NaPiIIa and NaPiIIc. Adaptation to a low Pi concentration for 24 h induces the expression of RNA from NaPiIIa and NaPiIIc, which is not prevented by actinomycin D. Small interfering RNA transfections revealed that PiT1 is not necessary for Pi transport, but it is necessary for adaptation to a low Pi, similar to NaPiIIa and PiT2. Our study reveals the complexity of the coordination between the four Pi transporters, the variability of RNA expression according to confluence and the heterogeneous correlation between Pi transport and RNA levels. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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29. Hypophosphatemia in vitamin D receptor null mice: effect of rescue diet on the developmental changes in renal Na-dependent phosphate cotransporters.
- Author
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Kaneko, Ichiro, Segawa, Hiroko, Furutani, Junya, Kuwahara, Shoji, Aranami, Fumito, Hanabusa, Etsuyo, Tominaga, Rieko, Giral, Hector, Caldas, Yupanqui, Levi, Moshe, Kato, Shigeaki, and Miyamoto, Ken-ichi
- Subjects
FAMILIAL hypophosphatemia ,VITAMIN D ,SODIUM cotransport systems ,PHOSPHATES ,DIET ,RICKETS ,LABORATORY mice ,BRUSH border membrane - Abstract
We analyzed vitamin D receptor (VDR) (−/−) mice fed either a normal diet or a rescue diet. Weanling VDR (−/−) mice had hypophosphatemia and hyperphosphaturia. Renal Na-dependent inorganic phosphate (Pi) cotransport activity was significantly decreased in weanling VDR (−/−) mice. In VDR (+/+) mice, renal Npt2a/Npt2c/PiT-2 protein levels were significantly increased at 21 and 28 days of age compared with that at 1 day of age. Npt2c and PiT-2 protein levels were maximally expressed at 28 days of age. Npt2a protein levels were significantly decreased in mice at 28 days of age compared with 21 and 60 days of age. In VDR (−/−) mice, Npt2a/Npt2c/PiT-2 protein levels were considerably lower than those in age-matched VDR (+/+) mice at 21 and 28 days of age. The reduced Npt2a/Npt2c/PiT-2 protein recovered completely in VDR-null mice fed the rescue diet. Although Pi transport activity and Npt2b were reduced in the proximal intestine in VDR (−/−) mice, Npt2b protein levels were not reduced in the distal intestine in VDR (−/−) mice. The rescue diet did not affect intestinal Npt2b protein levels in VDR (−/−) mice. Thus, reduced intestinal Pi absorption in VDR (−/−) mice does not seem to be the only factor that causes hypophosphatemia; reduced Npt2a, Npt2c, or PiT-2 protein levels during development might also cause hypophosphatemia and rickets in VDR (−/−) mice. Furthermore, dietary intervention completely normalized the expression of the renal phosphate transporters (Npt2a/Npt2c/PiT-2) in VDR (−/−) mice, suggesting that the lack of VDR activity is not the cause of impaired renal phosphate reabsorption. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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30. Differential regulation of the renal sodium-phosphate cotransporters NaPi-Ila, NaPi-IIc, and PiT-2 in dietary potassium deficiency.
- Author
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Breusegem, Sophia Y., Takahashi, Hideaki, Giral-Arnal, Hector, Wang, Xiaoxin, Jiang, Tao, Verlander, Jill W., Wilson, Paul, Miyazaki-Anzai, Shinobu, Sutherland, Eileen, Caldas, Yupanqui, Blame, Judith T., Segawa, Hiroko, Miyamoto, Ken-ichi, Barry, Nicholas P., and Levi, Moshe
- Subjects
PHOSPHATES ,SODIUM cotransport systems ,BRUSH border membrane ,POTASSIUM deficiency diseases ,KIDNEYS ,LABORATORY rats ,LABORATORY mice - Abstract
Dietary potassium (K) deficiency is accompanied by phosphaturia and decreased renal brush border membrane (BBM) vesicle sodium (Na)-dependent phosphate (P
i ) transport activity. Our laboratory previously showed that K deficiency in rats leads to increased abundance in the proximal tubule BBM of the apical Na-Pi cotransporter NaPi-Ila, but that the activity, diffusion, and clustering of NaPi-Ila could be modulated by the altered lipid composition of the K-deficient BBM (Zajicek HK, Wang H, Puttaparthi K, Halaihel N, Markovich D, Shayman J, Beliveau R, Wilson P, Rogers T, Levi M. Kidney mt 60: 694-704, 2001; Inoue M, Digman MA, Cheng M, Breusegem SY, Halaihel N, Sorribas V. Mantulin WW, Gratton E, Barry NP, Levi M. J Biol Chem 279: 49160-49171, 2004). Here we investigated the role of the renal Na-P cotransporters NaPi-lIc and PiT-2 in K deficiency. Using Western blotting, immunofluorescence, and quantitative real-time PCR, we found that, in rats and in mice, K deficiency is associated with a dramatic decrease in the NaPi-lIc protein abundance in proximal tubular BBM and in NaPi-lIc mRNA. In addition, we documented the presence of a third Na-coupled P, transporter in the renal BBM, PiT-2, whose abundance is also decreased by dietary K deficiency in rats and in mice. Finally, electron microscopy showed subcellular redistribution of NaPi-lIc in K deficiency: in control rats, NaPi-lIc immunolabel was primarily in BBM microvilli, whereas, in K-deficient rats, NaPiTIc BBM label was reduced, and immunolabel was prevalent in cytoplasmic vesicles. In summary, our results demonstrate that decreases in BBM abundance of the phosphate transporter NaPi-lIc and also PiT-2 might contribute to the phosphaturia of dietary K deficiency, and that the three renal BBM phosphate transporters characterized so far can be differentially regulated by dietary perturbations. [ABSTRACT FROM AUTHOR]- Published
- 2009
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31. Interaction of MAP17 with NHERF3/4 induces translocation of the renal Na/Pi ha transporter to the trans-Golgi.
- Author
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Lanaspa, Miguel A., Girai, Héctor, Breusegem, Sophia Y., Halaíhel, Nabil, Baile, Goretti, Catalán, Julia, Carrodeguas, José A., Barry, Nicholas P., Levi, Moshe, and Sorribas, Victor
- Subjects
GENE transfection ,PHOSPHATES ,CHROMOSOMAL translocation ,NEUROTRANSMITTERS ,PROTEIN kinases - Abstract
The function of the NaPiIIa renal sodium-phosphate transporter is regulated through a complex network of interacting proteins. Several PDZ domain-containing proteins interact with its COOH terminus while the small membrane protein MAP17 interacts with its NH2 end. To elucidate the function of MAP17, we identified its interacting proteins using both bacterial and mammalian two-hybrid systems. Several PDZ domain-containing proteins, including the four NHERF proteins, as well as NaPiIIa and NHE3, were found to bind to MAP17. The interactions of MAP17 with the NHERF proteins and with NaPiIIa were further analyzed in opossum kidney (OK) cells. Expression of MAP17 alone had no effect on the NaPiIIa apical membrane distribution, but coexpression of MAP17 and NHERF3 or NHERF4 induced internalization of NaPiIIa, MAP17, and the PDZ protein to the trans-Golgi network (TGN). This effect was not observed when MAP17 was cotransfected with NHERF1/2 proteins. Inhibition of protein kinase C (PKC) prevented expression of the three proteins in the TGN. Activation of PKC in OK cells transfected only with MAP17 induced complete degradation of MAPI7 and NaPiIIa. When lysosomal degradation was prevented, both proteins accumulated in the TGN. When the dopamine D1-like receptor was activated with fenoldopam, both NaPiIIa and MAP17 also accumulated in the TGN. Finally, cotransfection of MAP 17 and NHERF3 prevented the adaptive upregulation of phosphate transport activity in OK cells in response to low extracellular phosphate. Therefore, the interaction between MAP17, NHERF3/4, and NaPiIIa in the TGN could be an important intermediate or alternate path in the internalization of NaPiIIa. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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32. Post-transplant hypophosphatemia.
- Author
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Levi, Moshe
- Subjects
- *
FAMILIAL hypophosphatemia , *PHOSPHATES - Abstract
Discusses the causes, consequences and treatment of post-transplant hypophosphatemia. Regulation of serum phosphate concentration and renal phosphate transport; Potential mediators of post-transplant hypophosphatemia; Consequences and treatment of the disorder.
- Published
- 2001
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33. Late-onset downregulation of NaPi-2 in experimental Fanconi syndrome.
- Author
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Haviv, Yosef S., Wald, Hanna, Levi, Moshe, Dranitzki-Elhalel, Michal, and Popovtzer, M. M.
- Subjects
VETERINARY nephrology ,SODIUM cotransport systems ,PHOSPHATES ,GLOMERULAR filtration rate ,MALEIC acid ,PHYSIOLOGY - Abstract
The pathogenesis of renal phosphate (Pi) leak in Fanconi syndrome is unknown. Disorders of apical membrane transporters, leaky apical membrane, depleted cellular Pi and ATP, and impaired sodium (Na) pumps have been proposed as underlying defects. The present study examined the role of type II Na-Pi cotransport system (NaPi-2) in experimental Fanconi syndrome in rats. Following a single injection of maleic acid (MA), 75 mg/kg body weight IP, rats were sacrificed after 90 min, 4 h, and 24 h. Renal cortical expression of NaPi-2 mRNA was determined by Northern blotting, and brush border membrane (BBM) NaPi-2 protein by Western blotting. Increased urinary excretion of phosphate was demonstrated as soon as 90 min after MA injection, and was sustained at 4 and 24 h. NaPi-2 mRNA expression and NaPi-2 protein were not decreased after 90 min. NaPi-2 mRNA decreased after 4 h, while NaPi-2 protein decreased only at 24 h. Hence, the immediate phosphaturia in experimental Fanconi syndrome may be independent of NaPi-2 downregulation, possibly resulting from energy depletion or membrane dysfunction. The decrease in NaPi-2 mRNA expression and the subsequent NaPi-2 protein decrease may account for the second-phase phosphaturia. [ABSTRACT FROM AUTHOR]
- Published
- 2001
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34. Renal tubular sites of increased phosphate transport and NaPi-2 expression in the juvenile rat.
- Author
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Woda, Craig, Mulroney, Susan E., Halaihel, Nabil, Sun, Lijun, Wilson, Paul V., Levi, Moshe, and Haramati, Aviad
- Subjects
KIDNEY tubules ,PHOSPHATES ,ABSORPTION - Abstract
Presents information on a study which determined the tubular sites and mechanisms involved in renal phosphate reabsorption. Methodology; Results of the study; Discussion.
- Published
- 2001
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35. Cellular mechanisms of acute and chronic adaptation of rat renal Pi transporter to alterations in...
- Author
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Levi, Moshe and Lotscher, Marius
- Subjects
- *
PHOSPHATES , *KIDNEY physiology , *PHYSIOLOGY - Abstract
Discusses cellular mechanisms of acute and chronic adaptation of rat renal phosphate transporter to alterations in dietary phosphate. Sodium-phosphate cotransport measurements; Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting; RNA isolation; Chronic adaptation to low-phosphate diet.
- Published
- 1994
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36. Phosphonoformic acid blunts adaptive response of renal and intestinal Pi transport.
- Author
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Loghman-Adham, Mahmoud and Levi, Moshe
- Subjects
- *
PHOSPHONIC acids , *PHOSPHATES , *KIDNEY physiology , *BRUSH border membrane , *METABOLISM , *PHYSIOLOGY - Abstract
Assesses the effect of parenteral administration of phosphonoformic acid (PFA) on inorganic phosphate (Pi) handling. Effect of parenteral PFA on renal Pi excretion; Absorption by rat intestine; Effect of oral PFA on renal Pi excretion and plasma Pi; Effect of oral PFA on Pi transport in renal brush-border membrane vesicles (BBMV); Kinetics of effect of PFA in renal BBMV.
- Published
- 1993
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37. Toxicity of phosphonoformic acid in vascular smooth muscle cells: relationship to vascular calcification.
- Author
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Villa-Bellosta, Ricardo, Bogaert, Yolanda, Levi, Moshe, and Sorribas, Victor
- Subjects
PHOSPHATASES ,VASCULAR smooth muscle ,CALCIFICATION ,PHOSPHATES ,LACTATE dehydrogenase ,CELL death ,APOPTOSIS - Abstract
Medial vascular calcification (VC) develops as a consequence of hyperphosphatemia secondary to chronic kidney disease. The role of inorganic phosphate (Pi) transporters in the pathogenesis of VC has been previously determined by interference of RNA and inhibition by phosphonoformic acid (PFA). We have found that inhibition of Pi transport by PFA in vascular smooth muscle cells (VSMC) from rat aorta showed a K
i of 2.6 mM, while the Km for Pi was 0.1 mM. VSMC express both type III Pi transporters, Pit1 and Pit2. When expressed in Xenopus laevis oocytes Pit1 and Pit2 were inhibited with PFA with Ki values of 2.7 and 4.6 mM, respectively, while they showed affinities of 0.1 and 0.2 mM. PFA at either 1 or 5 mM completely prevented calcification of VSMC induced with 2 mM phosphate for 5 days. Phase-contrast microscopy revealed that cells treated with either 1 or 5 mM PFA in either normal (1 mM) or high (2 mM) Pi concentration showed a different morphology and evidence of cell death. Cytotoxicity was analyzed as a function of cytosolic lactate dehydrogenase activity, revealing a mean lethal concentration (LC50 ) of 0.38 mM. Therefore the prevention calcification was not due to inhibition of Pi transport but to cytotoxicity of PFA that prevented trans-differentiation of VSMC into osteogenic-like cells. Further experiments have been developed to characterize the type of cell death, including apoptosis. [ABSTRACT FROM AUTHOR]- Published
- 2007
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38. Mechanisms of inhibition of renal phosphate transport by phosphonoformate and arsenate.
- Author
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Villa-Bellosta, Ricardo, Levi, Moshe, and Sorribas, Victor
- Subjects
- *
PHOSPHATES , *ARSENATES , *CELLS , *HOMEOSTASIS , *PARATHYROID hormone , *CELL membranes - Abstract
Renal reabsorption of inorganic phosphate (Pi) is mediated through the activity of high affinity, type II Na-coupled Pi cotransporters in the proximal tubular cells. The rate of reabsorption is the main target for hormonal and metabolic factors that control phosphate homeostasis, including parathyroid hormone or adaptation to changes in dietary phosphate. Pi transport is also competitively inhibited by phosphonoformic acid (PFA) and arsenate, with Ki values of 0.13 and 0.2 mM, respectively, in opossum kidney cells (OK; an in vitro model of Pi renal reabsorption). However, NaPi4, the main type II Pi transporter in OK cells, is less efficiently inhibited by PFA and arsenate, with corresponding Ki values of 0.86 and 1 mM. Preincubation of OK cells with 1 mM PFA for 1 hour mimics acute adaptation to low Pi. However, preincubation with 1 mM arsenate has the same effect on transport activity as adaptation to 2 mM Pi. These effects are specific of Pi transport, as no alterations are observed in other Na-coupled transport processes including Na-glucose. Time-course and saturation kinetics suggest that these effects are mediated through changes in the abundance of Pi transporters in the plasma membrane. While both inhibitors act in a competitive manner, only arsenate is efficiently transported and, once inside the cell, it is also recognized as Pi by the sensing mechanisms that induce the cell to adapt to high Pi concentration. [ABSTRACT FROM AUTHOR]
- Published
- 2007
39. Liver X receptor-activating ligands modulate renal and intestinal sodium-phosphate transporters.
- Author
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Caldas, Yupanqui A., Giral, Hector, Cortázar, Michael A., Sutherland, Eileen, Okamura, Kayo, Blaine, Judith, Sorribas, Victor, Koepsell, Hermann, and Levi, Moshe
- Subjects
- *
CHOLESTEROL , *LIGANDS (Biochemistry) , *ATP-binding cassette transporters , *SODIUM , *PHOSPHATES , *LABORATORY mice - Abstract
Cholesterol is pumped out of the cells in different tissues, including the vasculature, intestine, liver, and kidney, by the ATP-binding cassette transporters. Ligands that activate the liver X receptor (LXR) modulate this efflux. Here we determined the effects of LXR agonists on the regulation of phosphate transporters. Phosphate homeostasis is regulated by the coordinated action of the intestinal and renal sodium-phosphate (NaPi) transporters, and the loss of this regulation causes hyperphosphatemia. Mice treated with DMHCA or TO901317, two LXR agonists that prevent atherosclerosis in ApoE or LDLR knockout mice, significantly decreased the activity of intestinal and kidney proximal tubular brush border membrane sodium gradient-dependent phosphate uptake, decreased serum phosphate, and increased urine phosphate excretion. The effects of DMHCA were due to a significant decrease in the abundance of the intestinal and renal NaPi transport proteins. The same effect was also found in opossum kidney cells in culture after treatment with either agonist. There was increased nuclear expression of the endogenous LXR receptor, a reduction in NaPi4 protein abundance (the main type II NaPi transporter in the opossum cells), and a reduction in NaPi co-transport activity. Thus, LXR agonists modulate intestinal and renal NaPi transporters and, in turn, serum phosphate levels. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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40. Role of PDZK1 Protein in Apical Membrane Expression of Renal Sodium-coupled Phosphate Transporters.
- Author
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Giral, Hector, Lanzano, Luca, Caldas, Yupanqui, Blaine, Judith, Verlander, Jill W., Tim Lei, Gratton, Enrico, and Levi, Moshe
- Subjects
- *
PROTEINS , *PHOSPHATES , *SODIUM , *PROTEIN-protein interactions , *KIDNEY tubules - Abstract
The sodium-dependent phosphate (Na/P1) transporters NaPi-2a and NaPi-2c play a major role in the renal reabsorption of P1. The functional need for several transporters accomplishing the same role is still not clear. However, the fact that these transporters show differential regulation under dietary and hormonal stimuli suggests different roles in P, reabsorption. The pathways controlling this differential regulation are still unknown, but one of the candidates involved is the NHERF family of scaffolding PDZ proteins. We propose that differences in the molecular interaction with PDZ proteins are related with the differential adaptation of Na/P1 transporters. Pdzkl-/- mice adapted to chronic low P1 diets showed an increased expression of NaPi-2a protein in the apical membrane of proximal tubules but impaired up-regulation of NaPi-2c. These results suggest an important role for PDZK1 in the stabilization of NaPi-2c in the apical membrane. We studied the specific protein-protein interactions of Na/P1 transporters with NHERF-1 and PDZK1 by FRET. FRET measurements showed a much stronger interaction of NHERF-1 with NaPi-2a than with NaPi-2c. However, both Na/P1 transporters showed similar FRET efficiencies with PDZK1. Interestingly, in cells adapted to low P1 concentrations, there were increases in NaPi-2c/PDZK1 and NaPi-2a/NHERF-1 interactions. The differential affinity of the Na/P1 transporters for NHERF-1 and PDZK1 proteins could partially explain their differential regulation and/or stability in the apical membrane. In this regard, direct interaction between NaPi-2c and PDZK1 seems to play an important role in the physiological regulation of NaPi-2c. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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41. Glycosphingolipids modulate renal phosphate transport in potassium deficiency.
- Author
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Zajicek, Hubert K., Wang, Huamin, Puttaparthi, Krishna, Halaihel, Nabil, Markovich, Daniel, Shayman, James, Béliveau, Richard, Wilson, Paul, Rogers, Thomas, and Levi, Moshe
- Subjects
- *
GLYCOSPHINGOLIPIDS , *PHOSPHATES , *POTASSIUM deficiency diseases - Abstract
Glycosphingolipids modulate renal phosphate transport in potassium deficiency. Background. Potassium (K) deficiency (KD) and/or hypokalemia have been associated with disturbances of phosphate metabolism. The purpose of the present study was to determine the cellular mechanisms that mediate the impairment of renal proximal tubular Na/Pi cotransport in a model of K deficiency in the rat. Methods. K deficiency in the rat was achieved by feeding rats a K-deficient diet for seven days, which resulted in a marked decrease in serum and tissue K content. Results. K deficiency resulted in a marked increase in urinary Pi excretion and a decrease in the Vmax of brush-border membrane (BBM) Na/Pi cotransport activity (1943 ± 95 in control vs. 1184 ± 99 pmol/5 sec/mg BBM protein in K deficiency, P < 0.02). Surprisingly, the decrease in Na/Pi cotransport activity was associated with increases in the abundance of type I (NaPi-1), and type II (NaPi-2) and type III (Glvr-1) Na/Pi protein. The decrease in Na/Pi transport was associated with significant alterations in BBM lipid composition, including increases in sphingomyelin, glucosylceramide, and ganglioside GM3 content and a decrease in BBM lipid fluidity. Inhibition of glucosylceramide synthesis resulted in increases in BBM Na/Pi cotransport activity in control and K-deficient rats. The resultant Na/Pi cotransport activity in K-deficient rats was the same as in control rats (1148 ± 52 in control + PDMP vs. 1152 ± 61 pmol/5 sec/mg BBM protein in K deficiency + PDMP). These changes in transport activity occurred independent of further changes in BBM NaPi-2 protein or renal cortical NaPi-2 mRNA abundance. Conclusion. K deficiency in the rat causes inhibition of renal Na/Pi cotransport activity by post-translational mechanisms that are mediated in part through alterations in glucosylceramide content and membrane lipid dynamics. [ABSTRACT FROM AUTHOR]
- Published
- 2001
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42. Gentamicin causes endocytosis of Na/Pi cotransporter protein (NaPi-2).
- Author
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Sorribas, Victor, Halaihel, Nabil, Puttaparthi, Krishna, Rogers, Thomas, Cronin, Robert E., Alcalde, Ana Isabel, Aramayona, Jose, Sarasa, Manuel, Huamin Wang, Wilson, Paul, Zajicek, Hubert, and Levi, Moshe
- Subjects
- *
SODIUM cotransport systems , *PHOSPHATES - Abstract
Determines the molecular mechanisms of impairment in sodium/phosphate cotransport activity. Decrease in superficial cortical brush-border membrane; Accumulation of sodium/phosphate protein; Colocalization of sodium/phosphate protein in endosomes.
- Published
- 2001
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- View/download PDF
43. Mechanisms of phosphate transport
- Author
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Carsten A. Wagner, Nati Hernando, Ian C. Forster, Victor Sorribas, Moshe Levi, Juerg Biber, Enrico Gratton, Heini Murer, University of Zurich, and Levi, Moshe
- Subjects
0301 basic medicine ,Fibroblast growth factor 23 ,PDZ domain ,030232 urology & nephrology ,610 Medicine & health ,Endocytosis ,Kidney ,Exocytosis ,10052 Institute of Physiology ,Phosphates ,03 medical and health sciences ,0302 clinical medicine ,Medicine ,Animals ,Humans ,Phosphate Transport Proteins ,2727 Nephrology ,business.industry ,Kidney metabolism ,Transporter ,Cell biology ,030104 developmental biology ,Nephrology ,570 Life sciences ,biology ,Kidney Diseases ,business ,Cotransporter ,Homeostasis - Abstract
Over the past 25 years, successive cloning of SLC34A1, SLC34A2 and SLC34A3, which encode the sodium-dependent inorganic phosphate (Pi) cotransport proteins 2a–2c, has facilitated the identification of molecular mechanisms that underlie the regulation of renal and intestinal Pi transport. Pi and various hormones, including parathyroid hormone and phosphatonins, such as fibroblast growth factor 23, regulate the activity of these Pi transporters through transcriptional, translational and post-translational mechanisms involving interactions with PDZ domain-containing proteins, lipid microdomains and acute trafficking of the transporters via endocytosis and exocytosis. In humans and rodents, mutations in any of the three transporters lead to dysregulation of epithelial Pi transport with effects on serum Pi levels and can cause cardiovascular and musculoskeletal damage, illustrating the importance of these transporters in the maintenance of local and systemic Pi homeostasis. Functional and structural studies have provided insights into the mechanism by which these proteins transport Pi, whereas in vivo and ex vivo cell culture studies have identified several small molecules that can modify their transport function. These small molecules represent potential new drugs to help maintain Pi homeostasis in patients with chronic kidney disease — a condition that is associated with hyperphosphataemia and severe cardiovascular and skeletal consequences. This Review describes the mechanisms by which dietary, hormonal and metabolic factors regulate the expression and function of sodium-dependent phosphate cotransporters. The authors discuss the consequences of dysregulated phosphate transport and how understanding of the structure–function relationships of the transporters provides insights into their transport mechanisms.
- Published
- 2019
44. Regulation of renal phosphate transport by acute and chronic metabolic acidosis in the rat.
- Author
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Ambühl, Patrice M., Zajicek, Hubert K., Wang, Huamin, Puttaparthi, Krishna, Levi, Moshe, and Wilson, the technical assistance of Paul
- Subjects
- *
ACIDOSIS , *PHOSPHATES , *ABSORPTION - Abstract
Regulation of renal phosphate transport by acute and chronic metabolic acidosis in the rat. Metabolic acidosis results in impaired renal tubular phosphate reabsorption and proximal tubular apical brush border membrane (BBM) sodium gradient-dependent phosphate transport (Na/Pi cotransport) activity. In the present study we investigated the cellular mechanisms responsible for decreased Na/Pi cotransport activity following six hours to 10 days of metabolic acidosis induced by ingestion of NH4 Cl. Urinary Pi excretion was significantly increased and BBM Na/Pi cotransport activity was progressively and significantly decreased by 18% at six hours, 24% at 12 hours, 32% at 24 hours, and 61% after 10 days of metabolic acidosis. The progressive and time-dependent decreases in BBM cotransport activity were associated with progressive decreases in BBM NaPi-2 protein (43% at 12 hr, 54% at 24 hr and 66% at 10 days) and cortical NaPi-2 mRNA (22% at 12 hr, 54% at 24 hr and 56% at 10 days) abundance. Interestingly, following six hours of metabolic acidosis, there was a significant 29% decrease in BBM NaPi-2 protein abundance that was not associated with decreases in either cortical homogenate NaPi-2 protein or cortical NaPi-2 mRNA abundance. In additional studies we found that the effects of chronic metabolic acidosis on Na/Pi cotransport activity were independent of endogenous parathyroid hormone activity, but were somewhat dependent on dietary Pi intake. In rats fed a high or a normal Pi diet metabolic acidosis caused significant decreases in Na/Pi cotransport activity, NaPi-2 protein and NaPi-2 mRNA abundance, however, in rats fed a low Pi diet the inhibitory effect of metabolic acidosis on Na/Pi cotransport were minimal and not significant. These results indicate that in chronic (≥ 12 hr) metabolic acidosis the progressive decrease in BBM Na/Pi cotransport activity is most likely mediated by decreases in BBM NaPi-2 protein and cortical mRNA abundance. In... [ABSTRACT FROM AUTHOR]
- Published
- 1998
- Full Text
- View/download PDF
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