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C H van Os

Publications and source records attributed to C H van Os.

At least 19 recordsLinked to original sources
PubMed2001

Functionality of aquaporin-2 missense mutants in recessive nephrogenic diabetes insipidus.

Aquaporin-2 (AQP2) missense mutants in recessive nephrogenic diabetes insipidus (NDI) are all retained in the endoplasmic reticulum (ER), but some could function as water channels. No conclusions could be drawn about the water permeability (Pf) of others, because there was no method for quantifying AQP2 expression in the plasma membrane. We recently developed such a method, which has allowed us to study the functionality of these AQP2 mutants. Immunoblot analysis of membranes of injected oocytes revealed that all mutants (AQP2-G64R, AQP2-N68S, AQP2 T126M, AQP2-A147T, AQP2-R187C, AQP2-S216P) are expressed as unglycosylated and high-mannose glycosylated AQP2. The level of the high-mannose form of AQP2-A147T in the plasma membranes was low, indicating that this mutation has a less severe effect on proper folding. Analysis of Pf values and plasma membrane expression levels reveals that AQP2-N68S, AQP2-R187C and AQP2-S216P are non-functional, AQP2-A147T is as functional as wt-AQP2, while AQP2-T126M and AQP2-G64R retain 20% of the permeability of wt-AQP2. Since G64 is highly conserved between AQPs and expected to form essential interactions with other amino acids within AQP1, the residual functionality of AQP2-G64R is surprising. Our data furthermore indicate that an eventual therapy with chemical chaperones that restores the routing of AQP2 mutants to the apical membrane of collecting ducts cells might relieve NDI in patients encoding AQP2-A147T, and to a lesser extent AQP2-T126M and AQP2-G64R, but not in patients encoding AQP2-N68S, AQP2-R187C or AQP2-S216P.

Amino Acid Sequence↗
PubMed2000

The subcellular localization of an aquaporin-2 tetramer depends on the stoichiometry of phosphorylated and nonphosphorylated monomers.

In renal principal cells, vasopressin regulates the shuttling of the aquaporin (AQP)2 water channel between intracellular vesicles and the apical plasma membrane. Vasopressin-induced phosphorylation of AQP2 at serine 256 (S256) by protein kinase A (PKA) is essential for its localization in the membrane. However, phosphorylated AQP2 (p-AQP2) has also been detected in intracellular vesicles of noninduced principal cells. As AQP2 is expressed as homotetramers, we hypothesized that the number of p-AQP2 monomers in a tetramer might be critical for the its steady state distribution. Expressed in oocytes, AQP2-S256D and AQP2-S256A mimicked p-AQP2 and non-p-AQP2, respectively, as routing and function of AQP2-S256D and wild-type AQP2 (wt-AQP2) were identical, whereas AQP2-S256A was retained intracellularly. In coinjection experiments, AQP2-S256A and AQP2-S256D formed heterotetramers. Coinjection of different ratios of AQP2-S256A and AQP2-S256D cRNAs revealed that minimally three AQP2-S256D monomers in an AQP2 tetramer were essential for its plasma membrane localization. Therefore, our results suggest that in principal cells, minimally three monomers per AQP2 tetramer have to be phosphorylated for its steady state localization in the apical membrane. As other multisubunit channels are also regulated by phosphorylation, it is anticipated that the stoichiometry of their phosphorylated and nonphosphorylated subunits may fine-tune the activity or subcellular localization of these complexes.

Animals↗
PubMed2000

Gene structure and chromosomal mapping of human epithelial calcium channel.

The epithelial Ca(2+) channel, ECaC, represents the rate-limiting step of vitamin D(3)-regulated Ca(2+) (re)absorption in kidney and intestine, and provides, therefore, a new candidate gene for Ca(2+)-related disorders. To supply the basis for direct mutation analysis, we report here the structure of the human ECaC gene (ECAC1(2)). It consists of 16 exons spanning 25 kb with introns ranging from 98 to 8500 bp. The 5'-flanking region of ECAC1 contains four putative vitamin D(3)-responsive elements. At positions -92 and -13 transcription initiation sites were identified, but the former lacks the canonical TATA or CAAT boxes. ECAC1 was mapped to chromosome 7q35 by fluorescent in situ hybridization, reassigning a previous radiation hybrid mapping to 7q31.1-2. The gene of a recently identified rat intestine homologue of ECaC, named Ca(2+) transporter 1, was found juxtaposed to the ECaC gene, indicating that both genes are the products of evolutionary local gene duplication.

Amino Acid Sequence↗
PubMed2000

Phsyiological relevance of aquaporins: luxury or necessity?

Aquaporins are members of a large family of pore-forming intrinsic membrane proteins, the MIP family. Based on their permeability properties they are now further subdivided into aquaporins, with real water-selective pores, and aquaglyceroporins with slightly less selective pores. Aquaporins are expressed in a large variety of tissues throughout the body but in most situations it is not clear whether their presence is necessary for the proper physiological function of these tissues. This review focuses on recent insight into the physiological relevance of aquaporins gained from studying aquaporin knockout mouse models and from diseases, on new surprising findings related to gating and selectivity, and on the consequences of tetramerization for routing and the genetics of nephrogenic diabetes insipidus. The active fluid transport in proximal tubules and in salivary glands is seriously compromised by aquaporin deletion. This is in contrast to lung, airways and stomach, where active fluid transport proceeds unhindered in the face of greatly reduced water permeabilities due to aquaporin deletion. Therefore, aquaporins seem to be a necessity at extreme high rates of active fluid transport but appear to be more of a luxury at medium or low fluid transport rates.

Animals↗
PubMed2000

Dissociation between urine osmolality and urinary excretion of aquaporin-2 in healthy volunteers.

BACKGROUND: It has been suggested that urinary excretion of the vasopressin-dependent water channel of the kidney collecting duct, aquaporin-2 (AQP2), reflects renal vasopressin action and might be used clinically. It is unclear, however, what relation exists between urine osmolality and urinary excretion of AQP2 (UAQP2) and it is unknown whether UAQP2 is influenced by hyperosmolality of urine or tubular flow rates. METHODS: We measured urine osmolality and UAQP2 in healthy volunteers in various conditions: (i) overnight dehydration continued during the day, (ii) after infusion of 700 ml hypertonic saline (NaCl 2.5%), and (iii) after intranasal administration of 40 microg 1-desamino-8-D-arginine vasopressin (DDAVP). The last two tests were performed after water loading. In addition, a DDAVP test was performed, after administration of frusemide. RESULTS: After overnight dehydration, the urine osmolality increased from 888+/-18 to 1004+/-17 mosmol/kg during additional hours of thirsting, whereas UAQP2 doubled from 140+/-45 to 285+/-63 fmol AQP2/micromol creatinine. Infusion of hypertonic saline increased urine osmolality from 70+/-3 to 451+/-68 mosmol/kg, while UAQP2 remained almost zero. Urine osmolality increased from 101+/-17 to 860+/-30 mosmol/kg after administration of DDAVP, with a parallel increase in UAQP2 from 32+/-14 to 394+/-81 fmol AQP2/micromol creatinine. Pre-treatment with frusemide attenuated the increase in urine osmolality, but had no effect on UAQP2 after DDAVP. CONCLUSIONS: Our data demonstrate that a simple relationship between urine osmolality and UAQP2 does not exist. Therefore, random or once-only measurements of UAQP2 as an index of renal vasopressin action are not useful. In contrast, intranasal application of DDAVP resulted in a parallel rise in urine osmolality and UAQP2. Therefore this test might be useful in studying patients with urine concentration defects. The DDAVP-frusemide test revealed that the release of AQP2 into urine is not caused by hypertonicity of tubular fluid.

Adult↗
PubMed2000

Epithelial calcium channel: gate-keeper of active calcium reabsorption.

The epithelial calcium channel present in the apical membrane of 1,25-dihydroxyvitamin D3-responsive nephron segments represents the first member of a new family of calcium channels. This review covers the distinctive properties of this highly calcium-selective channel and highlights the implications for our understanding of the process of calcium reabsorption.

Amino Acid Sequence↗
PubMed1999

The epithelial calcium channel, ECaC, is activated by hyperpolarization and regulated by cytosolic calcium.

The recently cloned epithelial Ca(2+) channel, ECaC, which is expressed in the apical membrane of 1,25-dihydroxyvitamin D(3)-responsible epithelia, was characterized in Xenopus laevis oocytes by measuring the Ca(2+)-activated Cl(-) current which is a sensitive read-out of the Ca(2+) influx. ECaC-expressing oocytes responded to a voltage ramp with a maximal inward current of -2.1 +/- 0.3 microA at a holding potential of -99 +/- 1 mV. The inward current decreased progressively at less negative potentials and at +50 mV a small Ca(2+)-induced outward current was observed. The Ca(2+) influx-evoked current at a hyperpolarizing pulse to -100 mV displayed a fast activation followed by a rapid but partial inactivation. Loading of the oocytes with the Ca(2+) chelator BAPTA delayed the activation and blocked the inactivation of ECaC. When a series of brief hyperpolarizing pulses were given a significant decline in the peak response and subsequent plateau phase was observed. In conclusion, the distinct electrophysiological features of ECaC are hyperpolarization-dependent activation, Ca(2+)-dependent regulation of channel conductance and desensitization during repetitive stimulation.

Animals↗
PubMed1999

An impaired routing of wild-type aquaporin-2 after tetramerization with an aquaporin-2 mutant explains dominant nephrogenic diabetes insipidus.

Autosomal recessive and dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin, are caused by mutations in the aquaporin-2 (AQP2) gene. Missense AQP2 proteins in recessive NDI have been shown to be retarded in the endoplasmic reticulum, whereas AQP2-E258K, an AQP2 mutant in dominant NDI, was retained in the Golgi complex. In this study, we identified the molecular mechanisms underlying recessive and dominant NDI. Sucrose gradient centrifugation of rat and human kidney proteins and subsequent immunoblotting revealed that AQP2 forms homotetramers. When expressed in oocytes, wild-type AQP2 and AQP2-E258K also formed homotetramers, whereas AQP2-R187C, a mutant in recessive NDI, was expressed as a monomer. Upon co-injection, AQP2-E258K, but not AQP2-R187C, was able to heterotetramerize with wild-type AQP2. Since an AQP monomer is the functional unit and AQP2-E258K is a functional but misrouted water channel, heterotetramerization of AQP2-E258K with wild-type AQP2 and inhibition of further routing of this complex to the plasma membrane is the cause of dominant NDI. This case of NDI is the first example of a dominant disease in which the 'loss-of-function' phenotype is caused by an impaired routing rather than impaired function of the wild-type protein.

Animals↗
PubMed1999

Molecular identification of the apical Ca2+ channel in 1, 25-dihydroxyvitamin D3-responsive epithelia.

In mammals, the extracellular calcium concentration is maintained within a narrow range despite large variations in daily dietary input and body demand. The small intestine and kidney constitute the influx pathways into the extracellular Ca2+ pool and, therefore, play a primary role in Ca2+ homeostasis. We identified an apical Ca2+ influx channel, which is expressed in proximal small intestine, the distal part of the nephron and placenta. This novel epithelial Ca2+ channel (ECaC) of 730 amino acids contains six putative membrane-spanning domains with an additional hydrophobic stretch predicted to be the pore region. ECaC resembles the recently cloned capsaicin receptor and the transient receptor potential-related ion channels with respect to its predicted topology but shares less than 30% sequence homology with these channels. In kidney, ECaC is abundantly present in the apical membrane of Ca2+ transporting cells and colocalizes with 1,25-dihydroxyvitamin D3-dependent calbindin-D28K. ECaC expression in Xenopus oocytes confers Ca2+ influx with properties identical to those observed in distal renal cells. Thus, ECaC has the expected properties for being the gatekeeper of 1,25-dihydroxyvitamin D3-dependent active transepithelial Ca2+ transport.

Amino Acid Sequence↗
PubMed1999

Time-dependent regulation by aldosterone of the amiloride-sensitive Na+ channel in rabbit kidney.

The epithelial Na+ channel (ENaC) functions as the rate-limiting factor in aldosterone-regulated transcellular Na+ transport. In the study described here, the effect of aldosterone on ENaC mRNA levels, protein synthesis and benzamil-sensitive Na+ transport was investigated using primary cultures of immunodissected rabbit kidney connecting tubule and cortical collecting duct cells (CNT and CCD, respectively). After a lag time of 3 h, aldosterone caused transepithelial Na+ transport to increase, reaching maximal level of 260+/-44% after 16 h of incubation. The alpha, beta and gamma rabbit ENaC (rbENaC) mRNA levels, measured by semi-quantitative reverse transcriptase-polymerase chain reaction, were not changed by aldosterone during the first 3 h, but a twofold increase was apparent after 6 h; levels remained elevated for up to 16 h of incubation. Immunoprecipitation of [35S]methionine-labeled rbENaC revealed a rise in protein levels of the alpha and beta subunits, but the protein level of the gamma subunit remained constant. In conclusion, our data suggest that in rabbit CNT and CCD the early increase in Na+ transport caused by aldosterone is due to the activation or insertion of existing Na+ channels into the apical membrane, and that the late response is mediated by increased synthesis of the alpha and beta rbENaC subunits.

Aldosterone↗
PubMed1999

Hormone-stimulated Ca2+ transport in rabbit kidney: multiple sites of inhibition by exogenous ATP.

Exogenous ATP markedly reduced 1-desamino-8-D-arginine vasopressin (dDAVP)-stimulated Ca2+ transport and cAMP accumulation in primary cultures of rabbit connecting tubule and cortical collecting duct cells. Similarly, ATP inhibited the stimulatory effect of 8-bromo-cAMP. At first sight, this is in agreement with the "classic" concept that dDAVP exerts its stimulatory effect via cAMP. However, dDAVP-stimulated Ca2+ transport was markedly reduced by the protein kinase C (PKC) inhibitor chelerythrine, reported previously to inhibit the cAMP-independent pathway responsible for parathyroid hormone-, [Arg8]vasopressin-, PGE2-, and adenosine-stimulated Ca2+ transport. Chelerythrine also inhibited the increase in Ca2+ transport evoked by the cAMP-independent A1 receptor agonist N6-cyclopentyladenosine (CPA). Downregulation of phorbol ester-sensitive PKC isoforms by chronic phorbol ester treatment has been shown before to be without effect on hormone-stimulated Ca2+ transport, indicating that the chelerythrine-inhibitable pathway consists of a phorbol ester-insensitive PKC isoform. Here, this maneuver did not affect ATP inhibition of dDAVP-stimulated Ca2+ transport and cAMP formation, while abolishing ATP inhibition of CPA-stimulated Ca2+ transport. These findings show that ATP acts via 1) a phorbol ester-sensitive PKC isoform to inhibit hormonal stimulation of Ca2+ transport at the level of the chelerythrine-inhibitable pathway involving a phorbol ester-insensitive PKC isoform and 2) a phorbol ester-insensitive mechanism to inhibit V2 receptor-mediated concomitant activation of this pathway and adenylyl cyclase.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

An aquaporin-2 water channel mutant which causes autosomal dominant nephrogenic diabetes insipidus is retained in the Golgi complex.

Mutations in the aquaporin-2 (AQP2) water channel gene cause autosomal recessive nephrogenic diabetes insipidus (NDI). Here we report the first patient with an autosomal dominant form of NDI, which is caused by a G866A transition in the AQP2 gene of one allele, resulting in a E258K substitution in the C-tail of AQP2. To define the molecular cause of NDI in this patient, AQP2-E258K was studied in Xenopus oocytes. In contrast to wild-type AQP2, AQP2-E258K conferred a small increase in water permeability, caused by a reduced expression at the plasma membrane. Coexpression of wild-type AQP2 with AQP2-E258K, but not with an AQP2 mutant in recessive NDI (AQP2-R187C), revealed a dominant-negative effect on the water permeability conferred by wild-type AQP2. The physiologically important phosphorylation of S256 by protein kinase A was not affected by the E258K mutation. Immunoblot and microscopic analyses revealed that AQP2-E258K was, in contrast to AQP2 mutants in recessive NDI, not retarded in the endoplasmic reticulum, but retained in the Golgi compartment. Since AQPs are thought to tetramerize, the retention of AQP2-E258K together with wild-type AQP2 in mixed tetramers in the Golgi compartment is a likely explanation for the dominant inheritance of NDI in this patient.

Adult↗
PubMed1998

Cold preservation of isolated rabbit proximal tubules induces radical-mediated cell injury.

BACKGROUND: Reactive oxygen species (ROS) are involved in reperfusion injury after preservation. Recent studies in isolated endothelial cells and hepatocytes suggested the occurrence of ROS-mediated injury during the period of cold incubation. In the present study, formation of ROS and subsequent cell injury were studied in freshly isolated rabbit proximal tubules (PTs). METHODS: PTs were incubated in University of Wisconsin (UW) solution, Euro-Collins solution, or a modified Krebs-Henseleit buffer under aerobic conditions for up to 94 hr at 4 degrees C. ROS formation and cell death were assessed as lipid peroxidation (formation of thiobarbituric acid-reactive substances [TBARS]) and release of lactate dehydrogenase, respectively. The involvement of ROS was further investigated in UW solution using compounds that might interfere with ROS formation. In addition, tubules were studied under anaerobic conditions (gassing with 95% N2/5% CO2). RESULTS: Cold preservation of rabbit PTs in any of the solutions under aerobic conditions caused progressive lipid peroxidation and concomitant cell injury. Addition to UW solution of inhibitors of ROS formation, in particular 2,2'-dipyridyl, or removal of oxygen by gassing with 95% N2/5% CO2, prevented lipid peroxidation and protected rabbit PTs against cold injury. Both the nitric oxide (NO) synthase inhibitor L-NAME and dexamethasone, which blocks the inducible NO synthase, were ineffective. The cytoprotectant glycine affected neither TBARS formation nor lactate dehydrogenase release. CONCLUSIONS: Cold preservation of renal PTs under aerobic conditions caused cell injury even in the specially designed preservation solution UW. Cell injury is caused by iron-dependent, NO synthase-independent ROS formation.

Adenosine↗
PubMed1998

The exchange of functional domains among aquaporins with different transport characteristics.

Aquaporins are transmembrane proteins that contain six bilayer-spanning domains, connected by loops A to E. The hourglass model predicts that the conserved loops B and E are essential for the formation of the water pore. To test the importance of loops B and E in the determination of the transport characteristics, we exchanged loops B and/or E between AQP0, AQP2, and AQP3. Detailed functional, immunoblot and immunocytochemical analyses of expression in Xenopus oocytes revealed that six out of the nine chimeric aquaporin proteins were not functional, because of misrouting. AQP0 with loop E of AQP2 was not impaired in its routing and revealed a low water permeability equal to that of wild-type AQP0. AQP2 with loop B of AQP0 was also routed normally and gave a high water permeability, similar to that of wild-type AQP2. AQP0 with loops B and E of AQP2 (AQP0–2BE) did not result in an increase in water permeability and was partly misrouted. However, the plasma membrane expression was high enough to expect an increase in water permeability, as loops B and E of AQP2 confer AQP2’s water permeability to AQP0. Although it is unclear for the dual chimera (AQP0–2BE), the parental water permeabilities obtained in oocytes expressing AQP0 with loop E of AQP2 or AQP2 with loop B of AQP0 indicate that, besides loops B and E, other parts of the AQP protein are important in the determination of the characteristics of the channel.

Animals↗
PubMed1998

Effects of renal cytoprotective agents on erythrocyte membrane stability.

To elucidate potential mechanisms of ischemic renal injury, investigators often use drugs that interfere with specific pathological pathways and study their protective efficacy in in vitro models of ischemia, such as isolated renal proximal tubules subjected to hypoxia. However, the protective effects of certain drugs may depend on non-specific membrane-stabilizing properties. We have studied the effects of several drugs on membrane integrity using osmotic lysis of erythrocytes as a model system. Freshly isolated rabbit erythrocytes were subjected to a hypotonic shock, and the protective effects of various calcium channel blockers, phospholipase inhibitors, free fatty acids, the NO-synthase inhibitor L-NAME, the amino acid glycine and its receptor-analogue strychnine, and two chloride channel blockers were examined. Most agents protected erythrocytes against hypotonic hemolysis when added to the medium in the same concentration range as used in suspensions of hypoxic proximal tubules. Only the protective agents that proposedly act via a blockade of chloride influx (glycine, strychnine and the chloride channel blockers), did not attenuate hypotonic hemolysis. The erythrocyte hemolysis assay may provide an easy and rapid method to screen for non-specific membrane-stabilizing effects of potentially cytoprotective agents.

Animals↗
PubMed1998

Epithelial aquaporins.

The past year has brought significant advances in our understanding of the involvement of aquaporins in the regulation of water balance. Besides the identification of new mammalian aquaporins, highlights include the progress in our understanding of their cell-biological regulation and their roles in physiology and pathophysiology as deduced from natural and engineered knockout models.

Amino Acid Sequence↗
PubMed1998

Hypoxia decreases calcium influx into rat proximal tubules.

Renal ischemia results in adenosine triphosphate (ATP) depletion, particularly in cells of the proximal tubule (PT), which rely heavily on oxidative phosphorylation for energy supply. Lack of ATP leads to a disturbance in intracellular homeostasis of Na+, K+ and Cl-. Also, cytosolic Ca2+ levels in renal PTs may increase during hypoxia [1], presumably by a combination of impaired extrusion and enhanced influx [2]. However, Ca2+ influx was previously measured using radiolabeled Ca2+ and at varying partial oxygen tension [2]. We have now used to Mn2(+)-induced quenching of fura-2 fluorescence to study Ca2+ influx in individual rat PTs during normoxic and hypoxic superfusion. Normoxic Ca2+ influx was indeed reflected by the Mn2+ quenching of fura-2 fluorescence and this influx could be inhibited by the calcium entry blocker methoxyverapamil (D600; inhibition 50 +/- 2% and 35 +/- 3% for 10 and 100 mumol, respectively). La3+ completely blocked normoxic Ca2+ influx. Hypoxic superfusion or rat PTs did not induce an increase in Ca2+ influx, but reduced this influx to 79 +/- 3% of the normoxic control. We hypothesize that reducing Ca2+ influx during hypoxia provides the cell with a means to prevent cellular Ca2+ overload during ATP-depletion, where Ca2+ extrusion is limited.

Adenosine Triphosphate↗