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H Murer

Publications and source records attributed to H Murer.

At least 91 records · Page 5Linked to original sources
PubMed1998

Detection and quantitation of a sodium-dependent sulfate cotransporter (NaSi-1) by sandwich-type enzyme-linked immunosorbent assay.

The sodium-dependent sulfate transporter (NaSi-1) DNA has been recently identified from rat kidney cortex. The objective of this study was to develop a quantitative assay for the NaSi-1 transporter protein. The NaSi-1 antigen was prepared by fusion protein techniques following analysis of the primary sequence for antigenicity. Polyclonal and monoclonal antibodies against the NaSi-1 antigen were raised in rabbits and mice, respectively. The specificity of the raised antibodies was examined by Western analysis using brush-border membrane (BBM) and basolateral membrane (BLM) purified from rat kidney cortex. Both NaSi-1 polyclonal and monoclonal antibodies detected a 69-kDa protein in the BBM. Using the purified monoclonal antibody as the capture antibody and the polyclonal antibody as the detecting antibody, a simple and sensitive sandwich-type enzyme-linked immunosorbent assay was developed to quantitate NaSi-1 transporter protein levels in tissue. The specificity of the assay was examined using BBM, BLM and NaSi-1-transfected Madin-Darby canine kidney cells. The assay was capable of detecting NaSi-1 at levels as low as 6.58 fmol. The concentration of NaSi-1 transporter protein in crude membrane isolated from rat kidney cortex was 0.094+/-0.014 fmol/ microg protein (mean+/-SD of three preparations).

Animals↗
PubMed1998

IGF-I and vanadate stimulate Na/Pi-cotransport in OK cells by increasing type II Na/Pi-cotransporter protein stability.

Insulin-like growth factor (IGF)-I and vanadate increase Na-dependent phosphate (Na/Pi) cotransport in opossum kidney (OK) cells. To gain more information about the mechanisms by which IGF-I and vanadate stimulate Na/Pi-cotransport, we measured type II Na/Pi-cotransporter (NaPi-4) protein abundance by Western blot analysis and investigated the effects of protein synthesis and tyrosine kinase inhibitors. The key findings in the present studies are as follows. First, incubation in IGF-I (10(-8) M) and/or vanadate (10(-3) M) for 3 h led to a non-additive 1.4-fold increase in Na/Pi-cotransport activity which was paralleled by a 1.5- to 2-fold increase in NaPi-4 protein. Second, actinomycin D did not abolish the increase in Na/Pi-cotransport and cycloheximide did not prevent the IGF-I-induced increase in Na/Pi-cotransport and NaPi-4 protein. Third, among the protein kinase inhibitors tested, only staurosporine substantially reduced the stimulation of Na/Pi-cotransport. In conclusion, the stimulatory effect of IGF-I on Na/Pi-cotransport is paralleled by an increased expression of NaPi-4 protein that is independent of protein synthesis and therefore results from increased protein stability. The observation that IGF-I and/or vanadate lead to similar increases in Na/Pi-cotransport and NaPi-4 protein abundance provides further evidence that the stimulation of Na/Pi-cotransport by IGF-I and vanadate involves protein tyrosine phosphorylation of the same signalling molecules.

Animals↗
PubMed1998

The renal type II Na+/phosphate cotransporter.

A sodium-dependent phosphate transporter (type II Na/Pi-cotransporter) was isolated which is expressed in apical membranes of proximal tubules and exhibits transport characteristics similar as described for renal reabsorption of phosphate. Type II associated Na/Pi-cotransport is electrogenic and results obtained by electrophysiological measurements support a transport model having a stoichiometry of 3 Na+/HPO4=. Changes of transport such as by parathyroid hormone and altered dietary intake of phosphate correlate with changes of the number of type II cotransporters in the apical membrane. These data suggest that the type II Na/Pi-cotransporter represents the main target for physiological and pathophysiological regulation.

Absorption↗
PubMed1998

Chromosomal localization of two human genes involved in phosphate homeostasis: the type IIb sodium-phosphate cotransporter and stanniocalcin-2.

Extracellular phosphate concentrations are maintained by coordinated regulation of specific homeostatic mechanisms. A novel gene, the type IIb sodium-phosphate cotransporter (Npt2b), was recently cloned and is expressed within intestinal tissues, indicating that the transporter may be an important regulator of phosphate reabsorption. Another gene, human stanniocalcin-2 (STC2), was previously shown to decrease phosphate uptake into kidney cells in vitro. Because of the important role that STC2 may play in phosphate homeostasis, we considered the peptide hormone a candidate for the phosphate wasting disease autosomal dominant hypophosphatemic rickets (ADHR), previously localized to chromosome 12p13. The purpose of our study was to determine the chromosomal localization of human NPT2b and STC2. In the present work, NPT2b was localized to human chromosome 4p15-p16, and STC2 to 5q33-tel. Because STC2 did not map to 12p13, the hormone was excluded as the ADHR gene, however it should be considered a candidate for other diseases involving phosphate homeostasis.

Calcium↗
PubMed1998

A PDZ domain-containing protein with homology to Diphor-1 maps to human chromosome 1q21.

The novel gene, Diphor-1, was recently cloned from rat kidney and shown to increase phosphate uptake in cells when co-expressed with a Na(+)-Pi cotransporter, indicating that it may play a substantial role in cellular phosphate balance. Previously, the phosphate wasting disorder, autosomal dominant hypophosphatemic rickets (ADHR) was mapped to chromosome 12p13 by linkage analysis. In the present work, PDZK1, a PDZ domain-containing protein highly homologous to rat Diphor-1, was shown to be expressed in human kidney. Based upon its sequence similarity to rat Diphor-1, we considered PDZK1 a feasible candidate gene for ADHR. PDZK1 was found to localize to human chromosome 1q21, thereby ruling it out as a candidate for ADHR.

Animals↗
PubMed1998

Parathyroid hormone and dietary phosphate provoke a lysosomal routing of the proximal tubular Na/Pi-cotransporter type II.

BACKGROUND: A decrease of proximal tubular reabsorption of phosphate (Pi), which can be provoked by parathyroid hormone (PTH) or by a high Pi-diet, has been shown to correlate with a decrease of the number of type II Na/Pi-cotransporters residing in the brush border membrane. While both PTH and a high Pi-diet lead to an internalization of type II cotransporters, the further cellular routing of internalized cotransporters has not been established unequivocally. METHODS: To prevent lysosomal degradation, rats were treated with leupeptin prior to the injection of PTH or feeding acutely with a high Pi-diet. Kidney cortex were recovered and used for immunohistochemistry. In parallel, brush border membranes and lysosomes were isolated and analyzed by Western blotting. RESULTS: Under both conditions (PTH and high Pi-diet), a strong overlap of internalized type II cotransporters with the late endosomes/lysosomes was observed by immunohistochemistry. In agreement, the content of type II Na/Pi-cotransporters was increased in lysosomes isolated from the corresponding tissues. CONCLUSIONS: These results suggest that in proximal tubular cells type II Na/Pi-cotransporters internalized due to the action of PTH and acute high Pi-diet are routed to the lysosomes, and likely do not enter a recycling compartment.

Animals↗
PubMed1998

The voltage dependence of a cloned mammalian renal type II Na+/Pi cotransporter (NaPi-2).

The voltage dependence of the rat renal type II Na+/Pi cotransporter (NaPi-2) was investigated by expressing NaPi-2 in Xenopus laevis oocytes and applying the two-electrode voltage clamp. In the steady state, superfusion with inorganic phosphate (Pi) induced inward currents (Ip) in the presence of 96 mM Na+ over the potential range -140 </= V </= +40 mV. With Pi as the variable substrate, the apparent affinity constant (KmPi) was strongly dependent on Na+, increasing sixfold for a twofold reduction in external Na+. KmPi increased with depolarizing voltage and was more sensitive to voltage at reduced Na+. The Hill coefficient was close to unity and the predicted maximum Ip (Ipmax) was 40% smaller at 50 mM Na+. With Na+ as the variable substrate, KmNa was weakly dependent on both Pi and voltage, the Hill coefficient was close to 3 and Ipmax was independent of Pi at -50 mV. The competitive inhibitor phosphonoformic acid suppressed the steady state holding current in a Na+-dependent manner, indicating the existence of uncoupled Na+ slippage. Voltage steps induced pre-steady state relaxations typical for Na+-coupled cotransporters. NaPi-2-dependent relaxations were quantitated by a single, voltage-dependent exponential. At 96 mM Na+, a Boltzmann function was fit to the steady state charge distribution (Q-V) to give a midpoint voltage (V0.5) in the range -20 to -50 mV and an apparent valency of approximately 0.5 e-. V0.5 became more negative as Na+ was reduced. Pi suppressed relaxations in a dose-dependent manner, but had little effect on their voltage dependence. Reducing external pH shifted V0.5 to depolarizing potentials and suppressed relaxations in the absence of Na+, suggesting that protons interact with the unloaded carrier. These findings were incorporated into an ordered kinetic model whereby Na+ is the first and last substrate to bind, and the observed voltage dependence arises from the unloaded carrier and first Na+ binding step.

Animals↗
PubMed1998

Characterization of the 5'-flanking region of OK cell type II Na-Pi cotransporter gene.

The renal type II Na-Pi cotransport is the rate-limiting step in proximal tubular phosphate (Pi) reabsorption. Among the different "proximal tubular" cell lines, this transporter seem only to be expressed in opossum kidney cells (OK cells). We have isolated the 5'-flanking region of the ok-Npt2 gene (OK cell type II Na-Pi cotransporter) including exons 1-3 and containing a TFIID site (TATA box), a GCCAAT site, an AP1 site, and two microsatellite GGAA repeats. Major transcription initiation sites were determined by primer extension and rapid amplification of 5' cDNA ends (5'-RACE). A 327-bp fragment containing the TFIID and GCAAT element was driving the downstream luciferase reporter gene in homologous transfection assays. Slightly reduced promoter activity was observed with a 198-bp fragment containing the GCAAT element; shorter fragments were without activity. Promoter activity (327-bp fragment) could also be observed in transfections into HeLa cells but not in U937 human macrophage cells, MCT mouse kidney cortex cells, and MDCK cells. Different "physiological" stimuli known to be associated with altered proximal tubular Na-Pi cotransport activity are without effect on transcriptional activity in above homologous transfection experiments.

Animals↗
PubMed1998

Immunolocalization of sat-1 sulfate/oxalate/bicarbonate anion exchanger in the rat kidney.

The rat liver sulfate/bicarbonate/oxalate exchanger (sat-1) transports sulfate across the canalicular membrane in exchange for either bicarbonate or oxalate. Sulfate/oxalate exchange has been detected in the proximal tubule of the kidney, where it is probably involved in the reabsorption of filtered sulfate and the secretion of oxalate and may contribute to oxalate-dependent chloride reabsorption. Screening of a renal cortex cDNA library determined that sat-1 is expressed in the rat kidney. To evaluate this anion exchanger, the sat-1 protein was expressed in Sf9 cells. Sodium-independent sulfate and oxalate uptake was enhanced 7.3-fold and 13.1-fold, respectively, in Sf9 cells expressing the sat-1 protein compared with cells infected with wild-type virus. We determined that sat-1 is glycosylated in the kidney; however, anion exchange via sat-1 is observed despite incomplete glycosylation of sat-1 in Sf9 cells. The sat-1 protein, with an added COOH-terminal 6-histidine tag, was purified on a metal affinity column and used to generate anti-sat-1 monoclonal antibodies. The sat-1 protein was localized to the basolateral membrane, but not the apical membrane, of the proximal tubule by both Western blot analysis and immunohistochemistry. These studies demonstrate that sulfate/oxalate exchange on the apical and basolateral membranes of the proximal tubule represents transport on two different anion exchangers.

Animals↗
PubMed1998

Renal Na/H exchanger NHE-3 and Na-PO4 cotransporter NaPi-2 protein expression in glucocorticoid excess and deficient states.

Administration of pharmacologic doses of glucocorticoid in vivo increases renal proximal tubule apical membrane Na/H exchange and decreases Na/PO4 cotransport activity (1). Current data suggest that the NHE-3 and NaPi-2 proteins mediate significant fractions of proximal tubule apical membrane Na/H exchange and Na/PO4 cotransport, respectively. This study examines whether glucocorticoid excess or deficiency affects NHE-3 and NaPi-2 protein abundance and the intrarenal distribution of these transporters. Protein abundance of NHE-3 and NaPi-2 in control rats was compared to rats rendered glucocorticoid-deficient by bilateral adrenalectomy, and to rats receiving pharmacologic doses of dexamethasone using immunoblots and immunohistochemistry. Adrenalectomy had modest effects on NHE-3 protein abundance, but dexamethasone administration to either adrenalectomized or sham-operated rats significantly increased NHE-3 protein abundance in both the proximal tubule and thick ascending limb, but not the thin descending limb. Adrenalectomy increased NaPi-2 protein abundance in the proximal tubule, whereas dexamethasone administration dramatically suppressed NaPi-2 protein on the apical membrane in both adrenalectomized and sham-operated animals. No significant reciprocal increase in subapical NaPi-2 staining was seen in the dexamethasone-treated rats. The present study shows that glucocorticoids regulate proximal tubule apical membrane Na/H exchange and NaPi cotransport by changes in protein abundance of NHE-3 and NaPi-2, respectively.

Adaptor Protein Complex 3↗
PubMed1998

Dietary sulfate regulates the expression of the renal brush border Na/Si cotransporter NaSi-1.

Dietary inorganic sulfate (Si) intake is an important factor in the regulation of renal proximal tubular sodium-dependent Si transport (Na/Si cotransport). The purpose of the present study was to determine whether modulation of Na/Si cotransport activity by dietary Si is mediated through regulation of the renal expression of the recently cloned NaSi-1 protein located in the apical brush border membrane (BBM) of the proximal tubule. It was found that rats fed a high Si diet had a marked increase in the renal excretion of Si and a concomitant decrease in BBM Na/Si cotransport activity when compared with rats on a control Si diet. The 43% decrease in BBM Na/Si cotransport activity was associated with a 33% decrease in BBM NaSi-1 protein abundance, as determined by Western blotting, and a 2.7-fold decrease in cortical NaSi-1 mRNA abundance, as determined by Northern blotting. Furthermore, cortical mRNA from rats fed a high Si diet when injected into Xenopus laevis oocytes led to a 2.2-fold decrease in Na/Si cotransport activity compared with mRNA isolated from control Si diet rats. This study indicates that adaptation to a high Si diet is accompanied by a decrease in renal cortical NaSi-1 mRNA abundance, which results in reduced expression of the NaSi-1 protein at the level of the proximal tubular BBM.

Adaptation, Physiological↗
PubMed1998

Cellular/molecular control of renal Na/Pi-cotransport.

A type II Na/Pi-cotransporter located in the brush border membrane is the rate limiting and physiologically regulated step in proximal tubular phosphate (Pi) reabsorption. In states of altered Pi-reabsorption [for example, in response to parathyroid hormone (PTH) and to altered dietary intake of Pi or as a consequence of genetic abnormalities], brush border expression of the type II Na/Pi-cotransporter is accordingly modified. PTH initiates a regulatory cascade leading to membrane retrieval, followed by lysosomal degradation of this transporter; recovery from inhibition requires its de novo synthesis. Pi-deprivation leads to an increased brush border expression of transporters that does not appear to require de novo synthesis in the short term. Pi-overload leads to membrane retrieval and degradation of transporters. Finally, in animals with genetically altered Pi-handling (Hyp; Gy) the brush border membrane expression of the type II Na/Pi-cotransporter is also reduced, suggesting that a genetically altered protein (such as PEX in Hyp) controls the expression of this transporter.

Animals↗
PubMed1998

Cellular mechanisms of renal adaptation of sodium dependent sulfate cotransport to altered dietary sulfate in rats.

The renal transport and fractional reabsorption of inorganic sulfate is altered under conditions of sulfate deficiency or excess. The objective of this study was to examine the cellular mechanisms of adaptation of renal sodium/sulfate cotransport after varying dietary intakes of a sulfur containing amino acid, methionine. Female Lewis rats were divided into four groups and fed diets containing various concentrations of methionine (0, 0.3, 0.82 and 2.46%) for 8 days. Urinary excretion rates and renal clearance of sulfate were significantly decreased in the animals fed a 0% methionine diet or a 0.3% methionine diet, and significantly increased in the animals fed a 2.46% methionine diet when evaluated on days 4 and 7. Serum sulfate concentrations were unchanged by diet treatment in all animals. The fractional reabsorption of sulfate was significantly increased in the animals fed the 0% methionine diet and the 0.3% methionine diets, and decreased in the animals fed the 2.46% methionine diet. Increased mRNA and protein levels for the sodium/sulfate transporter (NaSi-1) were found in the kidney cortex following treatment with the 0 and 0.3% methionine diet groups. Sulfate homeostasis by renal reabsorption is maintained by an up-regulation of steady state levels of NaSi-1 mRNA and protein when the diet is low in methionine.

Animals↗
PubMed1998

Ibuprofen-induced changes in sulfate renal transport.

Nonsteroidal anti-inflammatory drugs (NSAIDs) increase sulfate renal clearance and decrease the fractional reabsorption of sulfate by the kidneys. The mechanism of this alteration of inorganic sulfate homeostasis is unknown. The objectives of this study were 1) to investigate if sulfate renal transport is altered in isolated membrane vesicles after pretreatment of animals in vivo with ibuprofen (IBU), and 2) to determine the cellular mechanism of changes in sulfate renal transport. Female Lewis rats received IBU at a i.v. dose of 27 mg/kg followed by an infusion of 33 microg/min for 4 hr. Sulfate transport was studied using brush border (BBM) and basolateral membrane (BLM) vesicles isolated from rat kidney cortex. The Vmax for the sodium-dependent sulfate cotransport (NaSi-1) in BBM was significantly lower in the IBU group compared with the control group (0.79 +/- 0.23 vs. 1.25 +/- 0.17 nmol/mg protein/10 sec, respectively; P <.05) with no change in Km. There were no significant differences between the study groups in sulfate anion exchange kinetics in BLM vesicles. NaSi-1 transporter mRNA level in kidney cortex and protein level in BBM were significantly lower in animals pretreated with IBU compared with that in control animals. There was no change in membrane fluidity of BBM and BLM isolated from IBU-treated animals as measured by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. These results indicate that IBU treatment alters sodium-dependent sulfate cotransport by a downregulation of mRNA and protein of NaSi-1 transporter in BBM.

Animals↗
PubMed1997

Functional expression and purification of histidine-tagged rat renal Na/Phosphate (NaPi-2) and Na/Sulfate (NaSi-1) cotransporters.

Two mammalian sodium-dependent anion-cotransporters (NaPi-2 for phosphate and NaSi-1 for sulfate) have been expressed in Sf9 insect cells using the baculovirus expression system. A histidine tag was introduced at the C-termini in order to facilitate purification by metal-affinity chromatography. Sf9 cells infected with the histidine-tagged Ni/Pi-cotransporter exhibited more than 60-fold higher sodium-dependent transport of phosphate compared to noninfected cells. Expressed Na/Pi-cotransport exhibited a Km of Pi of 0.21 mm and an apparent Km of sodium of 92 mm. Infected cells expressed a 65 kDa polypeptide as detected by Western blotting and immunoprecipitation. Sf9 cells infected with the histidine-tagged NaSi-1 or untagged NaSi-1 protein expressed sodium-dependent sulfate cotransport up to 60-fold higher compared to noninfected cells. Transport of sulfate was highly dependent on sodium exhibiting a Km of SO2-4 of about 0.3-0.4 mm and a Km of sodium of 55 mm. By Western blotting and immunoprecipitation expressed NaSi-1 proteins were detected at 55-60 kDa. These studies demonstrate that histidine tagged proximal tubular Na-dependent cotransporters for phosphate and sulfate can be expressed functionally in Sf9 cells and that the kinetic characteristics were not altered by the introduction of a histidine tag at the C-termini. Furthermore, it is demonstrated that after solubilization under denaturing conditions histidine-tagged cotransporter proteins can be purified by metal-chelate affinity chromatography.

Animals↗
PubMed1997

Electrophysiological characterization of the flounder type II Na+/Pi cotransporter (NaPi-5) expressed in Xenopus laevis oocytes.

The two electrode voltage clamp technique was used to investigate the steady-state and presteady-state kinetic properties of the type II Na+/Pi cotransporter NaPi-5, cloned from the kidney of winter flounder (Pseudopleuronectes americanus) and expressed in Xenopus laevis oocytes. Steady-state Pi-induced currents had a voltage-independent apparent K(m) for Pi of 0.03 mM and a Hill coefficient of 1.0 at neutral pH, when superfusing with 96 mM Na+. The apparent K(m) for Na+ at 1 mM Pi was strongly voltage dependent (increasing from 32 mM at -70 mV to 77 mM at -30 mV) and the Hill coefficient was between 1 and 2, indicating cooperative binding of more than one Na+ ion. The maximum steady-state current was pH dependent, diminishing by 50% or more for a change from pH 7.8 to pH 6.3. Voltage jumps elicited presteady-state relaxations in the presence of 96 mM Na+ which were suppressed at saturating Pi (1 mM). Relaxations were absent in non-injected oocytes. Charge was balanced for equal positive and negative steps, saturated at extremes of potential and reversed at the holding potential. Fitting the charge transfer to a Boltzmann relationship typically gave a midpoint voltage (V0.5) close to zero and an apparent valency of approximately 0.6. The maximum steady-state transport rate correlated linearly with the maximum Pi-suppressed charge movement, indicating that the relaxations were NaPi-5-specific. The apparent transporter turnover was estimated as 35 sec-1. The voltage dependence of the relaxations was Pi-independent, whereas changes in Na+ shifted V0.5 to -60 mV at 25 mM Na+. Protons suppressed relaxations but contributed to no detectable charge movement in zero external Na+. The voltage dependent presteady-state behavior of NaPi-5 could be described by a 3 state model in which the partial reactions involving reorientation of the unloaded carrier and binding of Na+ contribute to transmembrane charge movement.

Animals↗