Different effects of arsenate and phosphonoformate on Pi transport adaptation in opossum kidney cells

2009 ◽  
Vol 297 (3) ◽  
pp. C516-C525 ◽  
Author(s):  
Ricardo Villa-Bellosta ◽  
Víctor Sorribas
Keyword(s):  
Rat Kidney ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Opossum Kidney Cells ◽  
Competitive Inhibitors ◽  
Renal Adaptation ◽  
Proximal Tubular ◽  
Dietary Phosphate ◽  
Definition Of

The main nonhormonal mechanism for controlling inorganic phosphate (Pi) homeostasis is renal adaptation of the proximal tubular Pi transport rate to changes in dietary phosphate content. Opossum kidney (OK) cell line is an in vitro renal model that maintains the ability of renal adaptation to the extracellular Pi concentration. We have studied how two competitive inhibitors of Pi transport, arsenate [As(V)] and phosphonoformate (PFA), affect adaptation to low and high Pi concentrations. OK cells show very high affinity for As(V) (inhibitory constant, Ki 0.12 mM) when compared with the rat kidney. As(V) very efficiently reversed the adaptation of OK cells to low Pi (0.1 mM), whereas PFA induced adaptation similar to 0.1 mM Pi. Adaptation with 2 mM Pi or As(V) was characterized by decreases in the maximal velociy ( Vmax) of Pi transport and an abundance of the NaPi-IIa Pi transporter in the plasma membrane, shown by the protein biotinylation. Conversely, PFA and 0.1 mM Pi increased the Vmax and transporter abundance. Changes in the Vmax were limited to a 50% variation, which was not paralleled by changes in the concentration of Pi or of the inhibitor. OK cells are very sensitive to As(V), but the effects are reversible and noncytotoxic. These effects can be interpreted as As(V) being transported into the cell, thereby mimicking a high Pi concentration. PFA blocks the uptake of Pi but is not transported, and it therefore simulates a low Pi concentration inside the cell. To conclude, a mathematical definition of the adaptation process is reported, thereby explaining the limited changes in Pi transport Vmax.

Autocrine/paracrine regulation of renal Na(+)-phosphate cotransport by dopamine

1993 ◽  
Vol 264 (4) ◽  
pp. F618-F622 ◽  
Author(s):  
R. P. Glahn ◽  
M. J. Onsgard ◽  
G. M. Tyce ◽  
S. L. Chinnow ◽  
F. G. Knox ◽  
...  
Keyword(s):  
In Vivo Model ◽  
Acid Decarboxylase ◽  
System Support ◽  
Opossum Kidney ◽  
Amino Acid Decarboxylase ◽  
Ok Cells ◽  
Proximal Tubular ◽  
Phosphate Cotransport

We tested the hypothesis that dopamine (DA) acts as an autocrine/paracrine regulator of Na(+)-Pi symport in proximal tubules, using opossum kidney (OK) cells as an in vivo model. Both DA and parathyroid hormone (PTH) increased adenosine 3',5'-cyclic monophosphate (cAMP) and inhibited Na(+)-gradient-dependent uptake of 32P but not that of L-[3H]-alanine. Incubation of OK cells with L-dopa, a DA precursor, resulted in accumulation of DA (7.4 nM), a ninefold increase of cAMP in the medium, and an inhibition (-10%) of Na(+)-Pi uptake. Carbidopa, an inhibitor of aromatic-L-amino acid decarboxylase, prevented the formation of DA from L-dopa, the increase in cAMP, and the inhibition of Na(+)-Pi cotransport. Pi-replete OK cells produced more DA (+15%) from L-dopa than Pi-deprived cells; however, the endogenous DA inhibited Na(+)-Pi cotransport both in Pi-deprived and in Pi-replete cells. Thus OK cells can synthesize DA from L-dopa in a quantity sufficient to elicit both the maximum DA-stimulated cAMP accumulation and inhibition of Na(+)-Pi cotransport in the same cell population. Our data, obtained on an in vitro system, support the hypothesis proposing that DA generated in proximal tubular cells can modulate, via cAMP, the Na(+)-Pi symport in the same or adjacent cells. If present in the kidney, this pathway might represent an autocrine/paracrine system that can contribute to regulation of renal Pi homeostasis.

scholarly journals Human munc13 Is a Diacylglycerol Receptor that Induces Apoptosis and May Contribute to Renal Cell Injury in Hyperglycemia

1999 ◽  
Vol 10 (5) ◽  
pp. 1609-1619 ◽  
Author(s):  
Yong Song ◽  
Menachem Ailenberg ◽  
Mel Silverman
Keyword(s):  
Phorbol Ester ◽  
Renal Cell ◽  
Cell Injury ◽  
Rat Kidney ◽  
Diabetic Rat ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Renal Cell Line ◽  
Diabetic Rat Model

We have previously shown that human munc13 (hmunc13) is up-regulated by hyperglycemia under in vitro conditions in human mesangial cell cultures. The purpose of the present study was to determine the cellular function of hmunc13. To do this, we have investigated the subcellular localization of hmunc13 in a transiently transfected renal cell line, opossum kidney cells. We have found that hmunc13 is a cytoplasmic protein and is translocated to the Golgi apparatus after phorbol ester stimulation. In addition, cells transfected with hmunc13 demonstrate apoptosis after treatment with phorbol ester, but cells transfected with an hmunc13 deletion mutant in which the diacylglycerol (C1) binding domain is absent exhibit no change in intracellular distribution and no induction of apoptosis in the presence of phorbol ester stimulation. We conclude that both the diacylglycerol-induced translocation and the apoptosis represent functional activity of hmunc13. We have also demonstrated that munc13-1 and munc13-2 are localized mainly to cortical epithelial cells in rat kidney and both are overexpressed under conditions of hyperglycemia in a streptozotocin-treated diabetic rat model. Taken together, our data suggest that hmunc13 serves as a diacylglycerol-activated, PKC-independent signaling pathway capable of inducing apoptosis and that this pathway may contribute to the renal cell complications of hyperglycemia.

Interactions of cubilin with megalin and the product of the amnionless gene (AMN): effect on its stability

2008 ◽  
Vol 410 (2) ◽  
pp. 301-308 ◽  
Author(s):  
Rajiv Ahuja ◽  
Raghunatha Yammani ◽  
Joseph A. Bauer ◽  
Seema Kalra ◽  
Shakuntla Seetharam ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Intrinsic Factor ◽  
Transmembrane Proteins ◽  
Immunohistochemical Analysis ◽  
Protein Product ◽  
Kidney Cells ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Polarized Epithelia

Cubilin, a 456 kDa multipurpose receptor lacking in both transmembrane and cytoplasmic domains is expressed in the apical BBMs (brush border membranes) of polarized epithelia. Cubilin interacts with two transmembrane proteins, AMN, a 45–50 kDa protein product of the amnionless gene, and megalin, a 600 kDa giant endocytic receptor. In vitro, three fragments of cubilin, the 113-residue N-terminus and CUB domains 12–17 and 22–27, demonstrated Ca2+-dependent binding to megalin. Immunoprecipitation and immunoblotting studies using detergent extracts of rat kidney BBMs revealed that cubilin interacts with both megalin and AMN. Ligand (intrinsic factor–cobalamin)-affinity chromatography showed that in renal BBMs, functional cubilin exists as a complex with both AMN and megalin. Cubilin and AMN levels were reduced by 80% and 55–60% respectively in total membranes and BBMs obtained from kidney of megalin antibody-producing rabbits. Immunohistochemical analysis and turnover studies for cubilin in megalin or AMN gene-silenced opossum kidney cells showed a significant reduction (85–90%) in cubilin staining and a 2-fold decrease in its half-life. Taken together, these results indicate that three distinct regions of cubilin bind to megalin and its interactions with both megalin and AMN are essential for its intracellular stability.

Use of phospho-specific antibodies to determine the phosphorylation of endogenous Na+/H+ exchanger NHE3 at PKA consensus sites

2005 ◽  
Vol 289 (2) ◽  
pp. F249-F258 ◽  
Author(s):  
Hetal S. Kocinsky ◽  
Adriana C. C. Girardi ◽  
Daniel Biemesderfer ◽  
Thao Nguyen ◽  
SueAnn Mentone ◽  
...  
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Rat Kidney ◽  
Subcellular Trafficking ◽  
Proximal Tubule Cells ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Type 3

Transfection studies using mutant constructs have implicated one or both protein kinase A (PKA) consensus phosphorylation sites [serines 552 and 605 in rat Na+/H+ exchanger type 3 (NHE3)] as critical for mediating inhibition of NHE3 in response to several stimuli including dopamine. However, whether one or both of these sites is actually phosphorylated in endogenous NHE3 in proximal tubule cells is unknown. The purpose of this study was to generate phosphospecific antibodies so that the state of phosphorylation of these serine residues in endogenous NHE3 could be assessed in vitro and in vivo. To this end, polyclonal and monoclonal phosphospecific peptide antibodies were generated against each PKA consensus site. Phosphospecificity was established by ELISA and Western blot assays. We then used these antibodies in vitro to evaluate the effect of dopamine on phosphorylation of the corresponding PKA sites (serines 560 and 613) in NHE3 endogenously expressed in opossum kidney cells. Baseline phosphorylation of both sites was detected that was significantly increased by dopamine. Next, we determined the baseline phosphorylation state of each serine in rat kidney NHE3 in vivo. We found that serine 552 of NHE3 is phosphorylated to a much greater extent than serine 605 at baseline in vivo. Moreover, we detected a distinct subcellular localization for NHE3 phosphorylated at serine 552 compared with total NHE3. Specifically, NHE3 phosphorylated at serine 552 localized to the coated pit region of the brush-border membrane, where NHE3 is inactive, while total NHE3 was found throughout the brush-border membrane. These findings strongly suggest that phosphorylation of NHE3 plays a role in its subcellular trafficking in vivo. In conclusion, we successfully generated phosphospecific antibodies that should be useful to assess the phosphorylation of endogenous NHE3 at its two PKA consensus sites under a variety of physiological conditions in vitro and in vivo.

scholarly journals Coexpression of MAST205 inhibits the activity of Na+/H+ exchanger NHE3

2006 ◽  
Vol 290 (2) ◽  
pp. F428-F437 ◽  
Author(s):  
Dongsheng Wang ◽  
Hye Jeong Lee ◽  
Deborah S. Cooper ◽  
Ludmila Cebotaro ◽  
Paul D. Walden ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Pdz Domain ◽  
Kinase Domain ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Apical Region ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Immunohistochemical Studies

Recent studies have shown that accessory proteins that interact with the apical Na+/H+ exchanger NHE3 are a vital part of the dynamic nature of the Na+/H+ exchanger regulation. We have identified MAST205, a microtubule-associated serine/threonine kinase with a molecular mass of 205 kDa that interacts with NHE3. MAST205 contains a S/T kinase domain and a PDZ domain that mediates interaction with NHE3. Northern blot analysis showed that MAST205 is highly expressed in human and rat kidney. Expression in opossum kidney (OK) cells showed that MAST205 is predominantly expressed in the apical membrane of the cells. Immunohistochemical studies demonstrated the presence of MAST205 at the apical region of the renal proximal tubules. Heterologous expression of MAST205 in OK cells inhibited endogenous NHE3 activity, and this inhibition required the presence of the kinase domain of MAST205, since deletion of the kinase domain or a dominant-negative mutant of MAST205 did not affect the activity of NHE3. Consistent with these results, we found that MAST205 phosphorylated NHE3 under in vitro conditions. However, overexpression of MAST205 did not affect expression of NHE3 proteins, suggesting that the effect of MAST205 was not mediated by a decrease in NHE3 expression. These findings suggest that MAST205 regulates NHE3 activity and, although the precise mechanism is yet to be determined, MAST205 appears to inhibit NHE3 activity through a phosphorylation-dependent mechanism.

Calcitriol stimulates Na(+)-Pi cotransport in a subclone of opossum kidney cells (OK-7A) by a genomic mechanism

1993 ◽  
Vol 264 (3) ◽  
pp. F404-F410 ◽  
Author(s):  
M. Allon ◽  
M. Parris
Keyword(s):  
Kidney Cells ◽  
Dihydroxyvitamin D3 ◽  
Prolonged Incubation ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Opossum Kidney Cells ◽  
Proximal Tubular ◽  
Renal Proximal Tubular Cells ◽  
Renal Proximal Tubular ◽  
Contrast Ct

Calcitriol (CT) stimulates Na(+)-Pi cotransport in a subclone of opossum kidney cells (OK-7A) by a genomic mechanism. An experimental model of renal proximal tubular cells in which CT affects Na(+)-Pi cotransport would be useful for examining the mechanisms of this effect. This study evaluated the effect of CT on Na(+)-Pi cotransport in opossum kidney (OK) cells. CT had no effect on Na(+)-Pi cotransport in wild-type OK cells and in the OK-B, OK-H, and OK-P subclones. In contrast, CT at physiological concentrations stimulated Na(+)-Pi cotransport in the OK-7A subclone; the effect was dose related with a 52% increase at 10(-11) M CT, as well as a maximal twofold stimulation at 10(-9) M. CT (10(-11) M) increased the maximum uptake for Na(+)-Pi cotransport (Vmax = 3.55 +/- 0.16 vs. 2.51 +/- 0.17 nmol.mg protein-1.5 min-1, P < 0.01), without affecting the apparent Michaelis constant (Km = 30.6 +/- 1.0 vs. 30.8 +/- 0.7 microM). The stimulatory effect on Na(+)-Pi cotransport was specific for CT and did not occur with 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, or 1 beta,25-dihydroxyvitamin D3. At 10(-11) M CT, the stimulation of Na(+)-Pi uptake in OK-7A cells was maximal at 3 h; it was completely abolished by preincubation with actinomycin D or cycloheximide. Both calphostin C, an inhibitor of protein kinase C (PKC), or prolonged incubation with phorbol 12-myristate 13-acetate, to downregulate the PKC pathway, partially inhibited the stimulatory effect of CT on Na(+)-Pi cotransport in OK-7A cells.(ABSTRACT TRUNCATED AT 250 WORDS)

NMR-visible intracellular P(i) and phosphoesters during regulation of Na(+)-P(i) cotransport in opossum kidney cells

1994 ◽  
Vol 267 (4) ◽  
pp. C915-C919 ◽  
Author(s):  
M. Barac-Nieto ◽  
A. Spitzer
Keyword(s):  
Brush Border Membrane ◽  
Maximum Velocity ◽  
Membrane Vesicles ◽  
Kidney Cells ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Opossum Kidney Cells ◽  
Igf I ◽  
Stimulation Of

There is an inverse relationship between intracellular concentration of P(i) ([P(i)]i) in the kidney and maximum velocity (Vmax) of Na(+)-P(i) cotransport in brush-border membrane vesicles both in P(i)-deprived and growing animals. However, at any given [P(i)]i, the Vmax is substantially higher in growing than in P(i)-deprived animals. This suggests that growth and P(i) depletion act on P(i) transport via different mechanisms. We tested this hypothesis by measuring the nuclear magnetic resonance-visible phosphate and the Vmax of Na(+)-P(i) cotransport in proximal tubule-like cells [opossum kidney (OK) cells] cultured in vitro. OK cells incubated in 1 mM extracellular P(i) had a [P(i)]i of 1.1 +/- 0.2 mM and a P(i) uptake of 1.47 +/- 0.06 nmol/mg in 5 min. Exposure of OK cells to P(i)-free medium decreased [P(i)]i by 80 +/- 7% (P < 0.01) and stimulated P(i) transport by 34 +/- 7% (P < 0.05). Exposure of OK cells to 10(-8) M insulin-like growth factor I (IGF-I) increased P(i) transport by 25 +/- 8% (P < 0.05) but did not affect [P(i)]i. The stimulation of Vmax produced by IGF-I was additive to that due to P(i) restriction. In addition, P(i) deprivation decreased the phosphomonoesters by 0.66 +/- 0.04-fold (P < 0.05) and increased the phosphodiesters by 2.5 +/- 0.5-fold (P < 0.01). Treatment with IGF-I increased both the phosphomonoesters (1.2 +/- 0.1-fold) and the phosphodiesters (4.1 +/- 0.6-fold). These results support the assumption that low P(i) supply and IGF-I stimulate Na(+)-P(i) cotransport by independent mechanisms.

scholarly journals Interaction of a farnesylated protein with renal type IIa Na/Pi co-transporter in response to parathyroid hormone and dietary phosphate

2004 ◽  
Vol 377 (3) ◽  
pp. 607-616 ◽  
Author(s):  
Mikiko ITO ◽  
Sachi IIDAWA ◽  
Michiyo IZUKA ◽  
Sakiko HAITO ◽  
Hiroko SEGAWA ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Rat Kidney ◽  
Intracellular Loop ◽  
Interacting Protein ◽  
Opossum Kidney ◽  
Opossum Kidney Cells ◽  
Dietary Phosphate ◽  
Dibasic Amino Acid ◽  
Farnesylated Protein ◽  
Hybrid Screening

Treatment with PTH (parathyroid hormone) or a high-Pi diet causes internalization of the type IIa sodium-dependent phosphate (Na/Pi IIa) co-transporter from the apical membrane and its degradation in the lysosome. A dibasic amino acid motif (KR) in the third intracellular loop of the co-transporter is essential for protein's PTH-induced retrieval. To elucidate the mechanism of internalization of Na/Pi IIa, we identified the interacting protein for the endocytic motif by yeast two-hybrid screening. We found a strong interaction of the Na/Pi IIa co-transporter with a small protein known as the PEX19 (human peroxisomal farnesylated protein; PxF, Pex19p). PEX19 can bind to the KR motif, but not to a mutant with this motif replaced with NI residues. PEX19 is highly expressed in mouse and rat kidney. Western blot analysis indicates that PEX19 is located in the cytosolic and brush-border membrane fractions (microvilli and the subapical component). Overexpression of PEX19 stimulated the endocytosis of the Na/Pi IIa co-transporter in opossum kidney cells in the absence of PTH. In conclusion, the present study indicates that PEX19 may be actively involved in controlling the internalization and trafficking of the Na/Pi IIa co-transporter.

Expression of the angiotensinogen gene is synergistically stimulated by 8-BrcAMP and Dex in opossum kidney cells

1995 ◽  
Vol 268 (1) ◽  
pp. R105-R111 ◽  
Author(s):  
M. Ming ◽  
T. T. Wang ◽  
S. Lachance ◽  
A. Delalandre ◽  
S. Carriere ◽  
...  
Keyword(s):  
Fusion Gene ◽  
Dependent Manner ◽  
Fusion Genes ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Angiotensinogen Gene ◽  
Opossum Kidney Cells ◽  
Dependent Protein ◽  
Flanking Region ◽  
Responsive Element

We transiently transfected fusion genes with the 5'-flanking region of the angiotensinogen gene linked to a bacterial chloramphenicol acetyltransferase (CAT) coding sequence as a reporter into opossum kidney (OK) cells. The addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) (10(-3)-10(-7) M) or forskolin (10(-9)-10(-5) M) stimulated the expression of the plasmid pOCAT [angiotensinogen nucleotide (N) -1498/+18] fusion gene in OK cells in a dose-dependent manner. The addition of dexamethasone (Dex) (10(-6) M) further enhanced the stimulatory effect of 8-BrcAMP or forskolin, whereas the addition of (R)-p-adenosine 3',5'-cyclic monophosphorothioate [(Rp)-cAMP[S], an inhibitor of cAMP-dependent protein kinase A, I and II] blocked the stimulatory effect of 8-BrcAMP. Furthermore, the addition of 8-BrcAMP (10(-3) M) or Dex (10(-6) M) or a combination of both stimulated the expression of pOCAT (angiotensinogen N -1138/+18), pOCAT (angiotensinogen N -960/+18), pOCAT (angiotensinogen N -814/+18), and pOCAT (angiotensinogen N -688/+18), but had no effect on the expression of pOCAT (angiotensinogen N -280/+18), pOCAT (angiotensinogen N -198/+18), pOCAT (angiotensinogen N -110/+18), pOCAT (angiotensinogen N -53/+18), and pOCAT (angiotensinogen N -35/+18). To further localize the putative cAMP-responsive element (CRE) in the angiotensinogen gene, we constructed fusion genes by inserting the DNA fragments angiotensinogen N -814 to N -689, angiotensinogen N -814 to N -761, and angiotensinogen N -760 to N -689 of the 5'-flanking region of the angiotensinogen gene upstream of the thymidine kinase (TK) promoter fused to a CAT gene and introduced them into OK cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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

1999 ◽  
Vol 276 (5) ◽  
pp. F720-F725 ◽  
Author(s):  
Markus F. Pfister ◽  
Jutka Forgo ◽  
Urs Ziegler ◽  
Jürg Biber ◽  
Heini Murer
Keyword(s):  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Phospholipase C ◽  
Type Ii ◽  
Peptide Fragments ◽  
Regulatory Pathways ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Kinase C ◽  
Proximal Tubular

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 β-12- O-tetradecanoylphorbol 13-acetate (β-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-Picotransport activity. Thereby, the cAMP-independent regulatory pathway leads only to partial effects (∼50%).

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