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N Hernando

Publications and source records attributed to N Hernando.

At least 19 recordsLinked to original sources
PubMed2006

Ifosfamide metabolites CAA, 4-OH-Ifo and Ifo-mustard reduce apical phosphate transport by changing NaPi-IIa in OK cells.

Renal Fanconi syndrome occurs in about 1-5% of all children treated with Ifosfamide (Ifo) and impairment of renal phosphate reabsorption in about 20-30% of them. Pathophysiological mechanisms of Ifo-induced nephropathy are ill defined. The aim has been to investigate whether Ifo metabolites affect the type IIa sodium-dependent phosphate transporter (NaPi-IIa) in viable opossum kidney cells. Ifo did not influence viability of cells or NaPi-IIa-mediated transport up to 1 mM/24 h. Incubation of confluent cells with chloroacetaldehyde (CAA) and 4-hydroperoxyIfosfamide (4-OH-Ifo) led to cell death by necrosis in a concentration-dependent manner. At low concentrations (50-100 microM/24 h), cell viability was normal but apical phosphate transport, NaPi-IIa protein, and -mRNA expression were significantly reduced. Coincubation with sodium-2-mercaptoethanesulfonate (MESNA) prevented the inhibitory action of CAA but not of 4-OH-Ifo; DiMESNA had no effect. Incubation with Ifosfamide-mustard (Ifo-mustard) did alter cell viability at concentrations above 500 microM/24 h. At lower concentrations (50-100 microM/24 h), it led to significant reduction in phosphate transport, NaPi-IIa protein, and mRNA expression. MESNA did not block these effects. The effect of Ifo-mustard was due to internalization of NaPi-IIa. Cyclophosphamide-mustard (CyP-mustard) did not have any influence on cell survival up to 1000 microM, but the inhibitory effect on phosphate transport and on NaPi-IIa protein was the same as found after Ifo-mustard. In conclusion, CAA, 4-OH-Ifo, and Ifo- and CyP-mustard are able to inhibit sodium-dependent phosphate cotransport in viable opossum kidney cells. The Ifo-mustard effect took place via internalization and reduction of de novo synthesis of NaPi-IIa. Therefore, it is possible that Ifo-mustard plays an important role in pathogenesis of Ifo-induced nephropathy.

Acetaldehyde↗
PubMed2006

Proximal tubular handling of phosphate: A molecular perspective.

Members of the SLC34 gene family of solute carriers encode for three Na+-dependent phosphate (P i) cotransporter proteins, two of which (NaPi-IIa/SLC34A1 and NaPi-IIc/SLC34A3) control renal reabsorption of P i in the proximal tubule of mammals, whereas NaPi-IIb/SCLC34A2 mediates P i transport in organs other than the kidney. The P i transport mechanism has been extensively studied in heterologous expression systems and structure-function studies have begun to reveal the intricacies of the transport cycle at the molecular level using techniques such as cysteine scanning mutagenesis, and voltage clamp fluorometry. Moreover, sequence differences between the three types of cotransporters have been exploited to obtain information about the molecular determinants of hormonal sensitivity and electrogenicity. Renal handling of P i is regulated by hormonal and non-hormonal factors. Changes in urinary excretion of P i are almost invariably mirrored by changes in the apical expression of NaPi-IIa and NaPi-IIc in proximal tubules. Therefore, understanding the mechanisms that control the apical expression of NaPi-IIa and NaPi-IIc as well as their functional properties is critical to understanding how an organism achieves P i homeostasis.

Animals↗
PubMed2005

NaPi-IIa and interacting partners.

Regulation of renal proximal tubular reabsorption of phosphate (Pi) is one of the critical steps in Pi homeostasis. Experimental evidence suggests that this regulation is achieved mainly by controlling the apical expression of the Na+-dependent Pi cotransporter type IIa (NaPi-IIa) in proximal tubules. Only recently have we started to obtain information regarding the molecular mechanisms that control the apical expression of NaPi-IIa. The first critical observation was the finding that truncation of only its last three amino acid residues has a strong effect on apical expression. A second major finding was the observation that the last intracellular loop of NaPi-IIa contains sequence information that confers parathyroid hormone (PTH) sensitivity. The use of the above domains of the cotransporter in yeast two-hybrid (Y2H) screening allowed the identification of proteins interacting with NaPi-IIa. Biochemical and morphological, as well as functional, analyses have allowed us to obtain insights into the physiological roles of such interactions, although our present knowledge is still far from complete.

Animals↗
PubMed2001

Amino acids involved in sodium interaction of murine type II Na(+)-P(i) cotransporters expressed in Xenopus oocytes.

Type IIa and IIb Na+-Pi cotransporters are highly conserved proteins expressed in brush border membranes of proximal tubules and small intestine, respectively. The kinetics of IIa and IIb differ significantly: type IIb is saturated at lower concentrations of Na+ and Pi. To define the domain responsible for the difference in Na+ affinity we constructed several mouse IIa-IIb chimeras as well as site-directed mutagenized cotransporters. Pi uptake activity was determined after injection of cRNAs into Xenopus laevis oocytes. From the chimera experiments we concluded that the domain containing part of the second intracellular loop, the fifth transmembrane domain (TD) and part of the third extracellular loop determines the specific Na+ activation properties for both types of cotransporter. Within this domain only a few residues located in the fifth TD are not conserved between type IIa and IIb. Site-directed mutagenesis on non-conserved residues was performed. Substitution of F402 of IIa by the corresponding L418 from IIb yielded a cotransporter that behaved like the IIb. On the other hand, substitution of the specific L418 of IIb by the corresponding F402 of IIa produced a cotransporter with a Na+ activation similar to IIa. (Single letter amino acid nomenclature is used throughout the paper.) These data suggest that the specific Na+ activation properties exhibited by type IIa and type IIb Na+-Pi cotransporters are at least in part due to the presence of a specific amino acid (F402 in IIa, and L418 in IIb) within the fifth TD of the protein.

Amino Acid Sequence↗
PubMed2001

Molecular determinants for apical expression of the renal type IIa Na+/Pi-cotransporter.

Type IIa and IIb Na+/Pi-cotransporters have different patterns of expression in vivo: IIa is expressed in apical membranes of renal proximal tubules, and IIb in intestinal and lung epithelia. They are found in different subcellular locations when transfected in epithelial cells: IIa is apically expressed in renal proximal cells (OK), but mostly intracellularly in intestinal cells (CaCo2); IIb is apical in both cell types. To identify the domains responsible for the different expression of both cotransporters (in CaCo2), as well as those responsible for the apical expression of IIa (in OK), mutated cotransporters were fused to the Enhanced Green Fluorescent Protein (EGFP), and their expression analyzed by confocal microscopy. We conclude that the apical expression information for CaCo2 is contained within the C-terminal tail of IIb, but is not contained within IIa. From analysis of mutated IIa cotransporters we identified residues, within the C-terminal tail, involved in the apical expression of these cotransporters in OK cells: internal PR-residues and terminal TRL-residues. These signals are functional in OK but not in CaCo2-cells, supporting the concept that polarized targeting can be protein and cell specific.

Amino Acid Motifs↗
PubMed2001

Regulation of the renal type IIa Na/Pi cotransporter by cGMP.

Inhibition of proximal tubular phosphate (Pi) reabsorption involves, as far as we know, brush border membrane retrieval of the type IIa Na/Pi-cotransporter. The aim of the present study was to analyze whether intracellular cGMP-mediated regulation of Pi reabsorption also involves retrieval of the type IIa Na/Pi-cotransporter, as previously shown for cAMP. Atrial natriuretic peptide (ANP) and nitric oxide (NO) were used to stimulate guanylate cyclase. In vivo perfusion of mice kidneys with either ANP or NO donors resulted in a downregulation of type IIa Na/Pi-cotransporters on the brush border membranes of proximal tubules. These effects were mimicked by activation of protein kinase G with 8Br-cGMP. In in-vitro-perfused mice proximal tubules, ANP was effective when added either to the apical or basolateral perfusate, suggesting the presence of receptors on both membrane sites. The effects of ANP and NO were blocked by the protein kinase G inhibitor LY 83553. Parallel experiments in OK cells, a renal proximal tubule model, provided similar information. Our findings document that cGMP-mediated regulation (ANP and NO) of type IIa Na/Pi-cotransporters also takes place via internalization of the transporter protein.

Animals↗
PubMed2001

Molecular determinants for apical expression and regulatory membrane retrieval of the type IIa Na/Pi cotransporter.

Renal inorganic phosphate (Pi) reabsorption is a key process in Pi homeostasis. Type IIa Na/Pi cotransporters, located at the apical membrane of renal proximal tubular cells, guarantee the vectorial transport of Pi. Renal Pi reabsorption can be modulated by controlling the number of cotransporters expressed at the apical membrane. Indeed, factors that increase Pi reabsorption induce the expression of type IIa cotransporters at the apical membrane, whereas factors that decrease Pi reabsorption lead to their retrieval. Therefore, proper sorting of this type of cotransporters is an essential step in Pi homeostasis. The relevance of polarization has been highlighted by the finding that improper sorting of transporters can cause disease. Here we describe the identification of signals involved in apical expression of newly synthesized type IIa cotransporters and in their hormonal-induced endocytosis.

Animals↗
PubMed2001

Molecular aspects in the regulation of renal inorganic phosphate reabsorption: the type IIa sodium/inorganic phosphate co-transporter as the key player.

The type IIa sodium/inorganic phosphate co-transporter is the rate-limiting inorganic phosphate transport pathway in renal brush-border membranes, and is thus a key player in overall inorganic phosphate homeostasis. Its regulation is mostly associated with membrane retrieval/reinsertion (traffic) of the transport protein. This membrane traffic is controlled by specific 'motifs' at the level of the transporter protein and probably involves interacting proteins (e.g. for scaffolding, regulation or sorting). The intracellular signaling mechanisms (e.g. the involvement of kinases) and the involvement of the cytoskeleton are not yet understood. Hereditary alterations in renal inorganic phosphate handling can be associated with factors controlling the expression of the brush-border type IIa sodium/inorganic phosphate co-transporter.

Absorption↗
PubMed2000

A dibasic motif involved in parathyroid hormone-induced down-regulation of the type IIa NaPi cotransporter.

Type II NaPi cotransporters are expressed in the apical membrane of P(i)-(re)absorbing epithelia: the type IIa in renal proximal tubule and the type IIb in small intestine. Parathyroid hormone (PTH) leads to a retrieval from the apical membrane of the type IIa NaPi cotransporter. The type IIa cotransporter is also expressed in opossum kidney (OK) cells, and its expression is under the control of PTH. In the present study, we identified the molecular "domains" involved in the PTH-induced retrieval of the type IIa NaPi cotransporter. Wild-type mouse type IIa (mIIa) and type IIb (mIIb) as well as several mIIa-mIIb chimeras and site-directed mutants were fused to the enhanced green fluorescent protein and transfected into OK cells. We found that mIIa but not mIIb was internalized and degraded after incubation with 1-34 (or 3-34) PTH. Using chimeras, we found that the N and C termini were not required in this effect, whereas a "domain" located between residues 216 and 658 seemed to be necessary. This region contains two putative intracellular loops with highly conserved sequences between mIIa and mIIb; in the last intracellular loop, two charged amino acids of type IIa (K(503)R(504)) are replaced by uncharged residues in type IIb (N(520)I(521)). We generated two mutants in which these residues were interchanged: mIIaNI and mIIbKR. Similarly to mIIa, the mIIbKR mutant was endocytosed in response to 1-34 PTH; in contrast, mIIaNI behaved as mIIb and was not internalized. In conclusion, a dibasic amino acid motif (K(503)R(504)) located in the last intracellular loop of the type IIa NaPi cotransporter is essential for its PTH-induced retrieval.

Amino Acid Motifs↗
PubMed2000

Requirement of a leucine residue for (apical) membrane expression of type IIb NaPi cotransporters.

Type II NaPi cotransporters mediate epithelial phosphate (P(i)) reabsorption. In mammals the type IIb protein is expressed in the small intestinal apical membrane and other epithelia; it is not expressed in the renal proximal tubule where we find the type IIa isoform. To look for molecular determinant(s) involved in apical expression of type IIb cotransporters, we have made deletion mutations within the C-terminal tails of mouse IIb (mIIb) and human IIb (hIIb) transporter proteins. The constructs were fused to the enhanced green fluorescent protein and transiently transfected into intestinal CaCo2-cells. Both mIIb and hIIb were located exclusively in the apical membrane of the cells. For mIIb, the removal of a cysteine cluster or the last three amino acids (TVF) had no effect on the location of the protein. However, truncation at the level of the conserved L691/689 prevented the apical membrane expression of both mIIb and hIIb, respectively, and the mutated proteins were located in endosomal and lysosomal structures. A similar expression pattern of the mIIb and hIIb constructs was found in renal proximal tubular opossum kidney cells. Our data suggest that L691/689 is involved in mechanisms leading to an apical expression of type IIb NaPi cotransporters.

Animals↗
PubMed2000

Molecular determinants of pH sensitivity of the type IIa Na/P(i) cotransporter.

Type II Na/P(i) cotransporters play key roles in epithelial P(i) transport and thereby contribute to overall P(i) homeostasis. Renal proximal tubular brush border membrane expresses the IIa isoform, whereas the IIb isoform is preferentially expressed in small intestinal brush border membrane of mammals. IIa and IIb proteins are predicted to contain eight transmembrane domains with the N- and C-terminal tails facing the cytoplasm. They differ in their pH dependences: the activity of IIa increases at higher pH, whereas the IIb shows no or a slightly opposite pH dependence. To determine the structural domains responsible for the difference in pH sensitivity, mouse IIa and IIb chimeras were constructed, and their pH dependence was characterized. A region between the fourth and fifth transmembrane domains was required for conferring pH sensitivity to the IIa-mediated Na/P(i) cotransport. Sequence comparison (IIa versus IIb) of the third extracellular loops revealed a stretch of three charged amino acids in IIa (REK) replaced by uncharged residues in IIb (GNT). Introduction of the uncharged GNT sequence (by REK) in IIa abolished its pH dependence, whereas introduction of the charged REK stretch in IIb (by GNT) led to a pH dependence similar to IIa. These findings suggest that charged residues within the third extracellular loop are involved in the pH sensitivity of IIa Na/P(i) cotransporter.

Amino Acid Sequence↗
PubMed2000

Asymmetrical targeting of type II Na-P(i) cotransporters in renal and intestinal epithelial cell lines.

Targeting of newly synthesized transporters to either the apical or basolateral domains of polarized cells is crucial for the function of epithelia, such as in the renal proximal tubule or in the small intestine. Recently, different sodium-phosphate cotransporters have been identified. Type II cotransporters can be subdivided into two groups: type IIa and type IIb. Type IIa is predominantly expressed in renal proximal tubules, whereas type IIb is located on the intestinal and lung epithelia. To gain some insights into the polarized targeting of the type II cotransporters, we have transiently expressed type IIa and type IIb cotransporters in several epithelial cell lines: two lines derived from renal proximal cells (opossum kidney and LLC-PK(1)), one from renal distal cells (Madin-Darby canine kidney), and one from colonic epithelium (CaCo-2). We studied the expression of the transporters fused to the enhanced green fluorescent protein. Our data indicate that the polarized targeting is dependent on molecular determinants most probably located at the COOH terminus of the cotransporters as well as on the cellular context.

Animals↗
PubMed2000

Proximal tubular phosphate reabsorption: molecular mechanisms.

Renal proximal tubular reabsorption of P(i) is a key element in overall P(i) homeostasis, and it involves a secondary active P(i) transport mechanism. Among the molecularly identified sodium-phosphate (Na/P(i)) cotransport systems a brush-border membrane type IIa Na-P(i) cotransporter is the key player in proximal tubular P(i) reabsorption. Physiological and pathophysiological alterations in renal P(i) reabsorption are related to altered brush-border membrane expression/content of the type IIa Na-P(i) cotransporter. Complex membrane retrieval/insertion mechanisms are involved in modulating transporter content in the brush-border membrane. In a tissue culture model (OK cells) expressing intrinsically the type IIa Na-P(i) cotransporter, the cellular cascades involved in "physiological/pathophysiological" control of P(i) reabsorption have been explored. As this cell model offers a "proximal tubular" environment, it is useful for characterization (in heterologous expression studies) of the cellular/molecular requirements for transport regulation. Finally, the oocyte expression system has permitted a thorough characterization of the transport characteristics and of structure/function relationships. Thus the cloning of the type IIa Na-P(i )cotransporter (in 1993) provided the tools to study renal brush-border membrane Na-P(i) cotransport function/regulation at the cellular/molecular level as well as at the organ level and led to an understanding of cellular mechanisms involved in control of proximal tubular P(i) handling and, thus, of overall P(i) homeostasis.

Absorption↗
PubMed1999

Effect of two tyrosine mutations on the activity and regulation of the renal type II Na/Pi-cotransporter expressed in oocytes.

The rat renal type II Na/Pi-cotransporter (NaPi2), which is regulated by mechanisms involving endocytosis and lysosomal degradation, contains two sequences that show high homology with two tyrosine (Y)-based consensus motifs previously reported to be involved in such intracellular trafficking: GY402FAM matching the consensus sequence GYXXZ, and Y509RWF matching the motif YXXO. Mutations of any of these two Y nearly abolished the NaPi2 mediated 32Pi-uptake after cRNA-injection into oocytes. The mechanisms underlying these defects are however different. Mutation of the Y402 results in a lack of glycosylation and reduced surface expression of the cotransporter, that are specific for the Y402 mutation since substitution of the neighboring F403 did not have any effect. The inhibitory effect of the Y509 mutation is related to a functional inactivation of the protein expressed in the plasma membrane; mutation of the neighboring R510 also led to a decrease in the cotransporter activity. Pharmacological activation of the protein kinase C cascade by DOG induced the retrieval of both wild-type (WT) as well as Y509 cotransporters from the oocyte plasma membrane. These data suggest that the Y402 is important for the surface expression whereas Y509 for the function of the type II Na/Pi-cotransporter expressed in oocytes. Y509 seems not to be involved in the membrane retrieval of the cotransporter.

Amino Acid Substitution↗
PubMed1999

Studies on the topology of the renal type II NaPi-cotransporter.

The rat type II sodium/phosphate cotransporter (NaPi-2) is a 85- to 90-kDa glycosylated protein located at the proximal tubular brush border membrane. Hydropathy predictions suggest eight transmembrane domains (sTM) with a large glycosylated loop between sTM 3 and sTM 4. We have studied the membrane topology of NaPi-2 expressed in oocytes. A 33-amino-acid fragment containing the FLAG epitope was inserted into seven loops connecting the sTMs and into the NH2- and COOH-ends of the protein. FLAG-antibody binding suggested that the loops connecting sTM 1 and sTM 2 as well as sTM 3 and sTM 4 are located extracellularly. Based on the lack of FLAG-antibody binding we suggest intracellular locations for the NH2- and COOH-termini and the region connecting sTM 4 and sTM 5. Immunoprecipitation studies of in vitro translated protein also suggest that the NH2-terminus is sited extracellularly. In immunohistochemical studies with NaPi-2-transfected MDCK cells, an interaction with NH2- and COOH- terminal antipeptide antibodies could only be obtained after membrane permeabilization. The presented data are an experimental documentation of the intracellular location of the NH2- and COOH-termini, and of the extracellular location of extracellular loops 1 and 2.

Amino Acid Sequence↗
PubMed1999

The presence of the alternatively spliced A2 cassette in the vacuolar H+-ATPase subunit A prevents assembly of the V1 catalytic domain.

Vacuolar ATPases (V-ATPases) are multisubunit enzymes that couple the hydrolysis of ATP to the transport of H+ across membranes, and thus acidify several intracellular compartments and some extracellular spaces. Despite the high degree of genetic and pharmacological homogeneity of V-ATPases, cells differentially modulate the lumenal pH of organelles and, in some cells, V-ATPases are selectively targetted to the plasma membrane. Although the mechanisms underlying such differences are not known, the subunit isoform composition of V-ATPases could contribute to altered assembly, targeting or activity. We previously identified an alternatively spliced variant of the chicken A subunit in which a 30 amino acid cassette (A1) containing the Walker consensus sequence for ATP binding is replaced by a 24 amino acid cassette (A2) that lacks this feature. We have examined the ability of chimeric yeast/chicken A subunits containing either the A1 or the A2 cassette to restore the V-ATPase activity of yeast that lack the A subunit. The A1-containing chimeric subunit, but not the chimera that contains the A2 cassette, partially restores the ability of the mutated yeast to grow at neutral pH. Both chimeric proteins are expressed, although at lower levels than the similarly transfected yeast A subunit. The A2-containing subunit fails to associate with the vacuolar membrane or support the assembly of V-ATPase complexes. Thus, the substitution of the A1 sequence by A2 not only removes the Walker nucleotide binding sequence but also compromises the ability of the A subunit to assemble with other V-ATPase subunits.

Adenosine Triphosphate↗