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J Biber

Publications and source records attributed to J Biber.

At least 73 records · Page 4Linked to original sources
PubMed1999

Rapid downregulation of rat renal Na/P(i) cotransporter in response to parathyroid hormone involves microtubule rearrangement.

Renal proximal tubule cells express in their apical brush border membrane (BBM) a Na/P(i) cotransporter type IIa that is rapidly downregulated in response to parathyroid hormone (PTH). We used the rat renal Na/P(i) cotransporter type IIa (NaPi-2) as an in vivo model to assess early cellular events in the rapid downregulation of this transporter. When rats were treated with PTH for 15 minutes, NaPi-2 abundance in the BBM was decreased. In parallel, transporter accumulated in intracellular vesicles. Concomitantly, microtubules (MTs) were found to form dense bundles of apical-to-basal orientation. After 60 minutes of PTH action, the cells were vastly depleted of NaPi-2, whereas their microtubular cytoskeleton had returned to its normal appearance. Prevention of MT rearrangement by taxol resulted in accumulation of NaPi-2 in the subapical cell portion after 15 minutes and a strong delay in depletion of intracellular transporter after 60 minutes of PTH action. Furthermore, the subapical accumulation of NaPi-2 was associated with the expansion of dense apical tubules of the subapical endocytic apparatus (SEA). Depolymerization of MTs by colchicine likewise caused a retardation of intracellular NaPi-2 depletion. These results suggest that NaPi-2 is downregulated in response to PTH through a rapid endocytic process in 2 separate steps: (a) internalization of the transporter into the SEA, and (b) its delivery to degradative organelles by a trafficking mechanism whose efficiency depends on a taxol-sensitive rearrangement of MTs.

Animals↗
PubMed1999

Role of thyroid hormone in regulation of renal phosphate transport in young and aged rats.

In the present study, we have examined the cellular mechanisms mediating the regulation of renal proximal tubular sodium-coupled inorganic phosphate (Na/Pi) transport by thyroid hormone (T3) in young and aged rats. Young hypothyroid rats showed a marked decrease in Na/Pi cotransport activity, which was associated with parallel decreases in type II Na/Pi cotransporter (NaPi-2) protein and messenger RNA (mRNA) abundance. In contrast, administration of long-term physiological and supraphysiological doses of T3 resulted in significant increases in Na/Pi cotransport activity, protein, and mRNA levels. Nuclear run-on experiments indicated that thyroid hormone regulates NaPi-2 mRNA levels by a transcriptional mechanism. In aged rats, although there were no changes in T3 serum levels (when compared with young animals), there were significant decreases in serum Pi concentration, renal Na/Pi cotransport activity, and NaPi-2 protein and mRNA abundance. These effects were mediated, at least in part, by a reduction in the transcriptional rate of the NaPi-2 gene, probably caused by, among other factors, a smaller response to the stimulatory action of T3. Compared with young rats, the old rats exhibited less sensitivity of the Na/Pi cotransporter to thyroid hormone, with-decreased effects in both hypothyroid (inhibitory) and hyperthyroid (stimulatory) animals.

Absorption↗
PubMed1999

Distribution of the sodium/phosphate transporter during postnatal ontogeny of the rat kidney.

Renal phosphate reabsorption via the type II sodium/ phosphate cotransporter (NaPi-2) in the brush border membrane (BBM) of proximal tubules underlies alterations during aging. The ontogeny of NaPi-2 in kidneys from newborn to 6-wk-old rats was investigated. NaPi-2 protein distribution in the kidneys of neonatal, 13-d-old, 22-d-old, and 6-wk-old rats was immunohistochemically analyzed, and NaPi-2 mRNA distribution in neonatal and 6-wk-old rats was analyzed by in situ hybridization. In kidneys of newborn rats, the appearance of NaPi-2 protein and mRNA coincided with the development of the brush border (assessed by actin staining) on proximal tubular cells. NaPi-2 was not detectable in the nephrogenic zone or in the outgrowing straight sections of proximal tubules, which lack a brush border. In 13-d-old suckling rats, strong NaPi-2 staining was seen in the BBM of convoluted proximal tubules of all nephron generations. In contrast, in 22-d-old weaned rats, NaPi-2 staining in the BBM of superficial nephrons was weaker than that in the BBM of juxtamedullary nephrons. Western blotting demonstrated that the overall abundance of NaPi-2 protein in the BBM of 22-d-old rats was decreased to approximately 70% of that in 13-d-old rats. In kidneys of 6-wk-old rats, the internephron gradient for NaPi-2 abundance in the BBM corresponded to that in adult rats. The data suggest that the NaPi-2 system in the kidney is fully functional and possesses the capacity for regulation as soon as nephrogenesis is completed. The manifestation of NaPi-2 internephron heterogeneity immediately after weaning might be related to the change in dietary inorganic phosphate content.

Animals↗
PubMed1998

Characterization of a murine type II sodium-phosphate cotransporter expressed in mammalian small intestine.

An isoform of the mammalian renal type II Na/Pi-cotransporter is described. Homology of this isoform to described mammalian and nonmammalian type II cotransporters is between 57 and 75%. Based on major diversities at the C terminus, the new isoform is designed as type IIb Na/Pi-cotransporter. Na/Pi-cotransport mediated by the type IIb cotransporter was studied in oocytes of Xenopus laevis. The results indicate that type IIb Na/Pi-cotransport is electrogenic and in contrast to the renal type II isoform of opposite pH dependence. Expression of type IIb mRNA was detected in various tissues, including small intestine. The type IIb protein was detected as a 108-kDa protein by Western blots using isolated small intestinal brush border membranes and by immunohistochemistry was localized at the luminal membrane of mouse enterocytes. Expression of the type IIb protein in the brush borders of enterocytes and transport characteristics suggest that the described type IIb Na/Pi-cotransporter represents a candidate for small intestinal apical Na/Pi-cotransport.

Amino Acid Sequence↗
PubMed1998

Preparation of basal cell membranes for scanning probe microscopy.

Scanning probe microscopy has the potential for investigating membranes in a physiological environment. We prepared with a lysis-squirting protocol basal cell membranes, that are suitable for scanning probe microscopy. Investigations using atomic force microscopy under liquid revealed cellular filaments which correlated perfectly with fluorescently stained actin filaments. Globular structures with a diameter as little as 10 nm could be resolved by stripping cytoplasmic components from the membranes. Therefore, cytoplasmic sides of supported basal cell membranes prove useful to gain high resolution with scanning probe microscopy in studies of plasma membrane associated structures and processes under buffer solution.

Animals↗
PubMed1998

[Molecular physiology and pathophysiology of renal phosphate excretion].

The kidney is a key organ in phosphate homeostasis. Phosphate excretion is largely determined by free glomerular filtration and by regulated reabsorption in the proximal tubule. The cellular/molecular mechanisms involved in phosphate reabsorption have been elucidated in great detail over the past few years. A brush border membrane protein with most probably 8 membrane-spanning regions represents the rate-limiting and physiologically/pathophysiologically modified transport mechanism. Altered phosphate reabsorption correlates with an altered brush border membrane transporter protein content, altered either by new synthesis/membrane insertion or by membrane retrieval/degradation. Current knowledge on the molecular/cellular level is a prerequisite for an understanding of kidney based alterations in phosphate homoeostasis.

Animals↗
PubMed1998

Chloride conductance and Pi transport are separate functions induced by the expression of NaPi-1 in Xenopus oocytes.

Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl- conductance (GCl), organic anion transport and Na+-dependent Pi-uptake. In the present study we investigated the relation between the NaPi-1 induced GCl and Pi-induced currents and transport. NaPi-1 expression induced Pi-transport, which was not different at 1-20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1-2 days after cRNA injection. In contrast, GCl was augmented at increased amounts of cRNA injection (1-20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. Pi-induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of Pi (>/= 1 mm Pi). The amplitudes of Ip correlated well with GCl. Ip was blocked by the Cl- channel blocker NPPB, partially Na+-dependent and completely abolished in Cl- free solution. In contrast, Pi-transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na+ and weakly affected by Cl- substitution. Endogenous Pi-uptake in water-injected oocytes amounted in all experiments to 30-50% of the Na+-dependent Pi-transport observed in NaPi-1 expressing oocytes. The properties of the endogenous Pi-uptake system (Km for Pi > 1 mM; partial Na+- and Cl--dependence; lack of NPPB block) were similar to the NaPi-1 induced Pi-uptake, but no Ip could be recorded at Pi-concentrations </=3 mM. In summary, the present data suggest that Ip does not reflect charge transfer related to Pi-uptake, but a Pi-mediated modulation of GCl.

Animals↗
PubMed1998

Parathyroid hormone leads to the lysosomal degradation of the renal type II Na/Pi cotransporter.

We have studied the involvement of proteolytic pathways in the regulation of the Na/Pi cotransporter type II by parathyroid hormone (PTH) in opossum kidney cells. Inhibition of lysosomal degradation (by leupeptin, ammonium chloride, methylamine, chloroquine, L-methionine methyl ester) prevented the PTH-mediated degradation of the transporter, whereas inhibition of the proteasomal pathway (by lactacystin) did not. Moreover it was found (i) that whereas lysosomal inhibitors prevented the PTH-mediated degradation of the transporter they did not prevent the PTH-mediated inhibition of the Na/Pi cotransport and (ii) that treating opossum kidney cells with lysosomal inhibitors led to an increased expression of the transporter without any concomitant increase in the Na/Pi cotransport. Further analysis by subcellular fractionation and morphological techniques showed (i) that the Na/Pi cotransporter is constitutively transported to and degraded within late endosomes/lysosomes and (ii) that PTH leads to the increased degradation of the transporter in late endosomes/lysosomes.

Acetylcysteine↗
PubMed1998

Cellular mechanisms involved in the acute adaptation of OK cell Na/Pi-cotransport to high- or low-Pi medium.

Variations in dietary phosphate (Pi) intake in rats lead to alterations of renal Pi reabsorption. These effects are associated with corresponding changes in the abundance of the type II Na/Pi-cotransporter protein in proximal tubular brush-border membranes. In the present study we investigated the regulation of the type II Na/Pi-cotransporter in response to high- and low-Pi medium in opossum kidney (OK) cells, an epithelial cell-line of proximal tubular origin. We show that "acute" (4 h) and "chronic" (24 h) exposures of OK cells to high- or low-Pi medium lead to decreases or increases, respectively, in Na/Pi-cotransport activity which are paralleled by alterations in the total cellular amount of the corresponding type II Na/Pi-cotransporter protein (NaPi-4), but not by changes in the amount of the NaPi-4 mRNA. Also in OK cells transfected with the corresponding rat renal type II Na/Pi-cotransporter (NaPi-2) alterations in the Pi concentration in the medium lead to changes in the amount of NaPi-2 protein but not in the amount of NaPi-2 mRNA. Furthermore we show that lysosomal inhibitors prevent the degradation of the transporter, but do not interfere with its inhibition, in response to "acute" exposure of OK cells to high-Pi medium. Inhibition of lysosomal degradation also leads, in control conditions, to an accumulation of the transporter detectable on Western blot. It is concluded that the lysosomal proteolytic pathway is not only involved in the Pi-induced downregulation of the type II Na/Pi-cotransporter but also in its basic turnover.

Adaptation, Physiological↗
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

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↗