Biomedical subjects

H Murer

Publications and source records attributed to H Murer.

At least 73 records · Page 4Linked to original sources

PubMed1999

Metabolic acidosis regulates rat renal Na-Si cotransport activity.

Recently, we cloned a cDNA (NaSi-1) localized to rat renal proximal tubules and encoding the brush-border membrane (BBM) Na gradient-dependent inorganic sulfate (Si) transport protein (Na-Si cotransporter). The purpose of the present study was to determine the effect of metabolic acidosis (MA) on Na-Si cotransport activity and NaSi-1 protein and mRNA expression. In rats with MA for 24 h (but not 6 or 12 h), there was a significant increase in the fractional excretion of Si, which was associated with a 2.4-fold decrease in BBM Na-Si cotransport activity. The decrease in Na-Si cotransport correlated with a 2.8-fold decrease in BBM NaSi-1 protein abundance and a 2.2-fold decrease in cortical NaSi-1 mRNA abundance. The inhibitory effect of MA on BBM Na-Si cotransport was also sustained in rats with chronic (10 days) MA. In addition, in Xenopus laevis oocytes injected with mRNA from kidney cortex, there was a significant reduction in the induced Na-Si cotransport in rats with MA compared with control rats, suggesting that MA causes a decrease in the abundance of functional mRNA encoding the NaSi-1 cotransporter. These findings indicate that MA reduces Si reabsorption by causing decreases in BBM Na-Si cotransport activity and that decreases in the expression of NaSi-1 protein and mRNA abundance, at least in part, play an important role in the inhibition of Na-Si cotransport activity during MA.

Acidosis↗

PubMed1999

Regulation of small intestinal Na-P(i) type IIb cotransporter by dietary phosphate intake.

Dietary restriction of phosphate is a well-known stimulator (acting indirectly via vitamin D(3)) of small intestinal apical Na-P(i) cotransport. In the present study, we document by Western blots and immunohistochemistry that, in mice, a low-P(i) diet given for several days leads (in parallel to a stimulation of Na-P(i) cotransport) to an increase of the abundance of the type IIb Na-P(i) cotransporter in the brush-border membrane of mouse enterocytes. Similar results were also obtained by an injection of cholecalciferol. The abundance of the type IIb transcript was investigated by Northern blots. These results indicated that the amount of the type IIb transcript was not changed by either low-P(i) diet or cholecalciferol. It is concluded that stimulation of intestinal Na-P(i) cotransport by low-P(i) diet and vitamin D(3) can be explained by an increased amount of type IIb Na-P(i) cotransporters in the brush-border membrane and that augmentation of type IIb Na-P(i) cotransporters is not related to an increased rate of transcription of the type IIb gene.

Animals↗

PubMed1999

Expression of type II Na-P(i) cotransporter in alveolar type II cells.

Type II Na-P(i) cotransporters (type IIa and type IIb) represent apically located Na-P(i) cotransporters in epithelia of proximal tubules (type IIa) and small intestine (type IIb). Here we provide evidence that the type IIb (but not the type IIa) Na-P(i) cotransporter is also expressed in the lung. With the use of immunohistochemistry, location of the type IIb protein was found exclusively in the apical membrane of type II cells of the alveolar epithelium. Such a location of the type IIb cotransporter suggests an involvement in the reuptake of phosphate necessary for the synthesis of surfactant. A possible regulation of the abundance of the type IIb cotransporter in the lung was studied after adaptation of mice to a low-P(i) diet. After a chronic adaptation to a low-P(i) diet, no changes in the type IIb protein and the type IIb transcript were observed. These results exclude dietary intake of phosphate as a regulatory factor of the type IIb Na-P(i) cotransporter in alveolar type II cells.

Adaptation, Physiological↗

PubMed1999

Effect of experimentally induced hypothyroidism on sulfate renal transport in rats.

Decreased serum sulfate concentrations are observed in hypothyroid patients. However, the mechanism involved in thyroid hormone-induced alterations of renal sulfate homeostasis is unknown. The objectives of this investigation were to determine the effect of 6-propyl-2-thiouracil (PTU)-induced hypothyroidism in rats on 1) the in vivo serum concentrations, renal clearance, and renal reabsorption of sulfate, 2) the in vitro renal transport in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, and 3) the cellular mechanism of the hypothyroid-induced alteration in sulfate renal transport. Serum sulfate concentrations, renal fractional reabsorption of sulfate, and creatinine clearance were decreased significantly in the hypothyroid group. The Vmax values for sodium-sulfate cotransport in BBM were significantly decreased in the kidney cortex from the hypothyroid animals (0.90 +/- 0.31 vs. 0.49 +/- 0.08 nmol. mg-1. 10 s-1, n = 5-6, P < 0.05) without changes in Km. There were no significant differences in Vmax and Km for sulfate/anion exchange transport in BLM. Sodium-dependent sulfate transporter (NaSi-1) mRNA and protein levels were significantly lower in the kidney cortex from hypothyroid rats. Hypothyroidism did not alter the membrane motional order (fluidity) in BBM and BLM, which indicates that the changes in the membrane fluidity do not represent the mechanism for the altered renal transport. These results demonstrate that PTU-induced hypothyroidism decreases sodium-sulfate cotransport by downregulation of the NaSi-1 gene.

Animals↗

PubMed1999

Type II Na-Pi cotransport is regulated transcriptionally by ambient bicarbonate/carbon dioxide tension in OK cells.

The purpose of the present study was to determine whether isohydric changes in HCO3 concentration and PCO2 directly affect apical Na-dependent Pi (Na-Pi) cotransport in OK cells (opossum kidney cell line). Cells were kept at either 44 mM NaHCO3/10% CO2, pH 7.4 (high-HCO3/CO2 condition), or 22 mM NaHCO3/5% CO2, pH 7.4 (low-HCO3/CO2 condition) (for 14-24 h). Incubation in lower HCO3/CO2 concentrations increased Na-Pi cotransport 1.5-fold. The increased Na-Pi cotransport was paralleled by a two- to threefold increased expression of the NaPi-4 transporter protein and a two- to threefold increase in NaPi-4 mRNA abundance. The increase in NaPi-4 mRNA could be completely prevented by incubation in the presence of a transcriptional inhibitor, suggesting that the increase in NaPi-4 mRNA results from an increased NaPi-4 mRNA transcription. In agreement, the NaPi-4 promoter activity was stimulated by 50% at lower HCO3/CO2 concentrations. In conclusion, our data demonstrate that isohydric changes in HCO3 concentration and PCO2 exert a significant, direct cellular effect on Na-Pi cotransport and NaPi-4 protein expression in OK cells by affecting NaPi-4 mRNA transcription.

Animals↗

PubMed1999

cAMP-dependent and -independent downregulation of type II Na-Pi cotransporters by PTH.

Parathyroid hormone (PTH) leads to the inhibition of Na-Pi cotransport activity and to the downregulation of the number of type II Na-Pi cotransporters in proximal tubules, as well as in opossum kidney (OK) cells. PTH is known also to lead to an activation of adenylate cyclase and phospholipase C in proximal tubular preparations, as well as in OK cells. In the present study, we investigated the involvement of these two regulatory pathways in OK cells in the PTH-dependent downregulation of the number of type II Na-Pi cotransporters. We have addressed this issue by using pharmacological activators of protein kinase A (PKA) and protein kinase C (PKC), i.e., 8-bromo-cAMP (8-BrcAMP) and beta-12-O-tetradecanoylphorbol 13-acetate (beta-TPA), respectively, as well as by the use of synthetic peptide fragments of PTH that activate adenylate cyclase and/or phospholipase C, i.e., PTH-(1-34) and PTH-(3-34), respectively. Our results show that PTH signal transduction via cAMP-dependent, as well as cAMP-independent, pathways leads to a membrane retrieval and degradation of type II Na-Pi cotransporters and, thereby, to the inhibition of Na-Pi cotransport activity. Thereby, the cAMP-independent regulatory pathway leads only to partial effects (approximately 50%).

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1999

Posttranscriptional regulation of the proximal tubule NaPi-II transporter in response to PTH and dietary P(i).

The rate of proximal tubular reabsorption of phosphate (P(i)) is a major determinant of P(i) homeostasis. Deviations of the extracellular concentration of P(i) are corrected by many factors that control the activity of Na-P(i) cotransport across the apical membrane. In this review, we describe the regulation of proximal tubule P(i) reabsorption via one particular Na-P(i) cotransporter (the type IIa cotransporter) by parathyroid hormone (PTH) and dietary phosphate intake. Available data indicate that both factors determine the net amount of type IIa protein residing in the apical membrane. The resulting change in transport capacity is a function of both the rate of cotransporter insertion and internalization. The latter process is most likely regulated by PTH and dietary P(i) and is considered irreversible since internalized type IIa Na-P(i) cotransporters are subsequently routed to the lysosomes for degradation.

Absorption↗

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↗

PubMed1999

Age- and growth hormone-induced alterations in renal sulfate transport.

The effects of growth hormone (GH) treatment on renal sodium sulfate cotransport (NaSi-1) were studied in adult (9-10 months) and old (22-23 months) male Fischer 344 rats. All animals received twice-daily s.c. injections of recombinant human GH (hGH; 4 mg/kg) for up to 6 days. Animals were sacrificed by exsanguination on days 0, 1, 2, 3, 4, 5, and 6. Kidneys were removed, and kidney cortex was trimmed immediately and used for RNA and membrane preparations. Plasma hGH concentrations were significantly lower in old rats during the hGH treatment (P <.05). Insulin-like growth factor-I (IGF-I) levels were significantly increased and remained stable after day 2 of hGH treatment in both age groups (P <.05). There was no significant difference in plasma IGF-I levels between age groups. Plasma IGF-I binding protein 3 (IGFBP-3) concentrations were significantly higher in 9- to 10-month-old rats compared with that in 22- to 23-month-old animals (P <.001). There were no significant differences in plasma IGFBP-3 concentrations between days of hGH treatment. The NaSi-1 mRNA levels were significantly lower in 22- to 23-month-old rats compared with that in 9- to 10-month-old animals (P <.001). The NaSi-1 mRNA levels were significantly increased on days 2 and 3 of hGH treatment (P <.05) and then gradually decreased to the control value. The NaSi-1 protein levels in old animals (22-23 months) were also significantly lower than that of 9- to 10-month-old animals and were significantly increased from day 2 of hGH treatment, reaching a maximum level on day 3 or 4 and then returning to the baseline level in both age groups. From these results, it was concluded that 1) NaSi-1 mRNA and protein levels are lower in old animals and increase in both adult and aged rats after hGH treatment, 2) plasma IGF-I levels are similar in adult and aged rats and increase after hGH treatment, and 3) plasma IGFBP-3 levels are lower in old rats and remain unchanged after hGH treatment.

Aging↗

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

Characterization of the human type II Na/Pi-cotransporter promoter.

The type II Na/Pi-cotransporter is expressed preferentially in renal proximal tubular epithelial cells. Comparison of the 5′ flanking region of the human NPT-2 gene with the opossum cell line (OK cell) and the murine Npt2 promoters revealed two conserved regions, one representing a putative C/EBP alpha site, the other a consensus TATA-box. In contrast to the OK cell and murine Npt-2 gene, the human exon 1 is flanked by two Alu-repeats, a short 90-bp inverted Alu element which is located within the promoter region and a full-length forward repeat present in intron 1. A 497-bp human promoter fragment including the inverted Alu-repeat was cloned in front of a luciferase reporter gene. The construct was active in OK and HeLa-S3 cells but no activity could be detected in the human monocyte cell line U937, the murine renal cortex cell line MCT and the dog kidney cell line MDCK. A twofold increase in promoter activity was observed in HeLa-S3 cells for a 5′ truncated fragment of 253 bp missing the inverted Alu-repeat. In the OK cell system the absence of the Alu-repeat was unable to modify promoter activity. In electrophoretic mobility shift assays (EMSAs) with a 31-bp oligonucleotide representing the conserved region with homology to C/EBP alpha we could provide evidence for specific DNA/protein interactions with nuclear extracts derived from kidney and liver cell lines but not for HeLa-S3 and U937 nuclear extracts. Specific interactions could also be observed with nuclear extracts from renal cortex, medulla and rat liver but not from rat spleen, intestine and heart. Southern-Western blotting techniques suggest that a 31-kDa nuclear protein from kidney-derived cells binds to the C/EBP-like region of the NPT2 promoter.

Animals↗