R.J.M. Bindels

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.

The Calcium-Sensing Receptor Promotes Urinary Acidification to Prevent Nephrolithiasis

Hypercalciuria increases the risk for urolithiasis, but renal adaptive mechanisms reduce this risk. For example, transient receptor potential vanilloid 5 knockout (TPRV5(-/-)) mice lack kidney stones despite urinary calcium (Ca(2+)) wasting and hyperphosphaturia, perhaps as a result of their significant polyuria and urinary acidification. Here, we investigated the mechanisms linking hypercalciuria with these adaptive mechanisms. Exposure of dissected mouse outer medullary collecting ducts to high (5.0 mM) extracellular Ca(2+) stimulated H(+)-ATPase activity. In TRPV5(-/-) mice, activation of the renal Ca(2+)-sensing receptor promoted H(+)-ATPase-mediated H(+) excretion and downregulation of aquaporin 2, leading to urinary acidification and polyuria, respectively. Gene ablation of the collecting duct-specific B1 subunit of H(+)-ATPase in TRPV5(-/-) mice abolished the enhanced urinary acidification, which resulted in severe tubular precipitations of Ca(2+)-phosphate in the renal medulla. In conclusion, activation of Ca(2+)-sensing receptor by increased luminal Ca(2+) leads to urinary acidification and polyuria. These beneficial adaptations facilitate the excretion of large amounts of soluble Ca(2+), which is crucial to prevent the formation of kidney stones.

Calbindin-D28K facilitates cytosolic calcium diffusion without interfering with calcium signaling

The role of calbindin-D28K, in transcellular Ca2+ transport and Ca2+ signaling in rabbit cortical collecting system was investigated. Rabbit kidney connecting tubules and cortical collecting ducts, hereafter referred to as cortical collecting system, were isolated by immunodissection and cultured to confluence on permeable filters and glass coverslips. Calbindin-D28K was present in the cytosol of principal cells, but was absent from the intercalated cells. 1,25(OH)2D3 (48 h, 10(-7) M) significantly increased cellular calbindin-D28K levels (194 +/- 15%) and stimulated transcellular Ca2+ transport (41 +/- 3%). This stimulatory effect could be fully mimicked by the endogenous Ca2+ chelator, BAPTA (30 microM BAPTA/AM), which suggests that the presence of Ca2+ chelators alone is sufficient to enhance transcellular Ca2+ transport. Stimulation of Ca2+ transport was not accompanied by a rise in [Ca2+]i. Isosmotic replacement of extracellular Na+ ([Na+]o) for N-methylglucamine (NMG) generated oscillations in [Ca2+]i in individual cells of the monolayer. The functional parameters of these oscillations such as frequency of spiking, resting [Ca2+]i, increase in [Ca2+]i and percentage of responding cells, were not affected by the level of calbindin-D28K. In contrast, loading the cells with BAPTA abruptly stopped these [Ca2+]i oscillations. This suggests that the kinetics of Ca2+ binding by calbindin-D28K are slow relative to the initiation of the [Ca2+]i rise, so that calbindin-D28K, unlike BAPTA, is unable to reduce [Ca2+]i rapidly enough to prevent the initiation of Ca(2+)-induced Ca2+ release.

Role of Na+/Ca2+ exchange in transcellular Ca2+ transport across primary cultures of rabbit kidney collecting system

Cells from connecting tubule and cortical collecting duct of rabbit kidney were isolated by immunodissection with mAb R2G9 and cultured on permeable filters. Confluent monolayers developed an amiloride-sensitive transepithelial potential difference of −50±1 mV (lumen negative) and a transepithelial resistance of 507±18 Ω cm2. Transepithelial Ca2+ transport increased dose-dependently with apical [Ca2+] and, in solutions containing 1 mM Ca2+, the active transcellular Ca2+ transport rate was 92±2 nmol h−1 cm−2. Transcellular Ca2+ transport was dependent on basolateral Na+ (Nab+). Isoosmotic substitution of Nab+for N-methylglucamine resulted in a concentration-dependent decrease in Ca2+ absorption, with maximal inhibition of 67±5%. A Hill plot of the Na+-dependence yielded a coefficient of 1.9±0.4, indicating more than one Na+ site on a Na+-dependent Ca2+ transport system. In addition, the absence of Cab2+resulted in a significant increase in Ca2+ transport both in the presence and absence of Nab+. Added basolaterally, ouabain (0.1 mM) inhibited Ca2+ transport to the same extent as did Na+-free solutions, while bepridil (0.1 mM), an inhibitor of Na+/Ca2+ exchange, reduced Ca2+ transport by 32±6%. Methoxyverapamil, felodipine, flunarizine and diltiazem (10 μM) were without effect. Depolarisation of the basolateral membrane, by raising [K+]b to 60 mM, significantly decreased transcellular Ca2+ transport, which is indicative of electrogenic Na+/Ca2+ exchange. In conclusion, active Ca2+ transport in the collecting system of rabbit kidney is largely driven by basolateral Na+/Ca2+ exchange. However, a residual Ca2+ absorption of about 30% was always observed, suggesting that other Ca2+ transport mechanisms, presumably a Ca2+-ATPase, participate as well.

Critical Role of the Epithelial Ca2+ Channel TRPV5 in Active Ca2+ Reabsorption as Revealed by TRPV5/Calbindin-D28K Knockout Mice

The epithelial Ca(2+) channel TRPV5 facilitates apical Ca(2+) entry during active Ca(2+) reabsorption in the distal convoluted tubule. In this process, cytosolic Ca(2+) remains at low nontoxic concentrations because the Ca(2+) influx is buffered rapidly by calbindin-D(28K). Subsequently, Ca(2+) that is bound to calbindin-D(28K) is shuttled toward the basolateral Ca(2+) extrusion systems. For addressing the in vivo role of TRPV5 and calbindin-D(28K) in the maintenance of the Ca(2+) balance, single- and double-knockout mice of TRPV5 and calbindin-D(28K) (TRPV5(-/-), calbindin-D(28K)(-/-), and TRPV5(-/-)/calbindin-D(28K)(-/-)) were characterized. These mice strains were fed two Ca(2+) diets (0.02 and 2% wt/wt) to investigate the influence of dietary Ca(2+) content on the Ca(2+) balance. Urine analysis indicated that TRPV5(-/-)/calbindin-D(28K)(-/-) mice exhibit on both diets hypercalciuria compared with wild-type mice. Ca(2+) excretion in TRPV5(-/-)/calbindin-D(28K)(-/-) mice was not significantly different from TRPV5(-/-) mice, whereas calbindin-D(28K)(-/-) mice did not show hypercalciuria. The similarity between TRPV5(-/-)/calbindin-D(28K)(-/-) and TRPV5(-/-) mice was supported further by an equivalent increase in renal calbindin-D(9K) expression and in intestinal Ca(2+) hyperabsorption as a result of upregulation of calbindin-D(9K) and TRPV6 expression in the duodenum. Elevated serum parathyroid hormone and 1,25-dihydroxyvitamin D(3) levels accompanied the enhanced expression of the Ca(2+) transporters. Intestinal Ca(2+) absorption and expression of calbindin-D(9K) and TRPV6, as well as serum parameters of the calbindin-D(28K)(-/-) mice, did not differ from those of wild-type mice. These results underline the gatekeeper function of TRPV5 being the rate-limiting step in active Ca(2+) reabsorption, unlike calbindin-D(28K), which possibly is compensated by calbindin-D(9K).

Apical and basolateral expression of Aquaporin-1 in transfected MDCK and LLC-PK cells and functional evaluation of their transcellular osmotic water permeabilities

Abstract Aquaporin-1 is present in the apical and basolateral membranes in proximal tubules and descending limbs of Henlé’s loop. In order to be able to study the routing of Aquaporin-1 and the regulation of Aquaporin-1-mediated transcellular water flow, we stably transfected LLC-PK1 and MDCK-HRS cell lines with an Aquaporin-1 expression construct. LLC-PK1 clone 7 and MDCK clone K integrated two and one copies, respectively, which was reflected in the amount of Aquaporin-1 mRNA expressed in both clones. The Aquaporin-1 protein levels, however, were similar. In both clones, immuno-electronmicroscopy showed extensive labelling of Aquaporin-1 on the basolateral plasma membrane, endosomal vesicles and the apical plasma membrane, including the microvilli. To measure transcellular water permeation, a simple method was applied using phenol-red as a cell-impermeant marker of concentration. In contrast to the native cell lines, both clones revealed a high transcellular osmotic water permeability, which could not be influenced by forskolin add/3-isobutyl-1-methylxanthine (IBMX) or the phorbol ester 12-O-tetradecanoyl 13-acetate (TPA). After glutaraldehyde fixation, it was inhibitable by HgCl2. These results indicate that targeting of Aquaporin-1 to the apical and basolateral plasma membrane is independent of cell type and show for the first time that water flow through a cultured epithelium can be blocked by mercurial compounds.

Assignment of the canalicular multispecific organic anion transporter gene (CMOAT) to human chromosome 10q24 and mouse chromosome 19D2 by fluorescent in situ hybridization

Rabbit epithelial basolateral chloride conductance regulator (EBCR) and rat canalicular multispecific organic anion transporter (Cmoat) are found to be homologues based on protein sequence comparison and Northern blot analysis. EBCRis, therefore, renamed as rabbit Cmoat. The gene encoding CMOAT, a transporter possibly involved in Dubin-Johnson syndrome in humans, is mapped on human chromosome 10q24 and mouse chromosome 19D2.

A modified confocal laser scanning microscope allows fast ultraviolet ratio imaging of intracellular Ca2+ activity using Fura-2

Abstract A confocal, ultraviolet laser scanning microscope (LSM) for reliable ratio measurements of localized intracellular Ca2+ gradients using the Ca2+-sensitive dye Fura-2 was developed. In a commercial LSM, the filter wheels for the excitation band-pass filters and the grey filters were replaced by acousto-optic tunable filters (AOTF) for rapid switching (≤1.5 μs) of the ultraviolet (351 and 364 nm) and the visible (457, 476, 488, 514 nm) excitation light. This enabled dual wavelength excitation of Fura-2, or 2’7’-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) for pH measurements. Changing to a transmitted-light detector of high sensitivity allowed for simultaneous recording of differential interference contrast images of the preparation with the excitation light. The AOTF fine control of the intensity of the excitation light and improvements in the emission detector sensitivity enabled the acquisition of up to 120 ratio pairs of high-quality images from a single cell. The optical capabilities and limitations of the instrument were evaluated with fluorescent beads and dye-loaded cultured cells. Agonist-induced intracellular Ca2+ transients in HT29 cells were recorded to test for the instrument’s ability to measure changes in [Ca2+]i. Ratio z-sections from Fura-2-loaded cells showed an inhomogeneity of the Fura-2 loading with an accumulation of the dye mostly in the mitochondria. We show, as an example of the microscope’s achievable resolution, the spatial and temporal heterogeneity of [Ca2+]i signals in mitochondria and the cytosol in response to agonist-evoked stimulation of HT29 cells. In addition, we show that the lipophilic, membrane-bound Fura-2 derivative Fura-C18, for measurements of near-membrane Ca2+ changes, can be used with this confocal microscope. This new LSM is expected to deepen our understanding of localized [Ca2+]i signals; for example, the nuclear Ca2+ signalling or the [Ca2+]i changes that occur during stimulation of ion secretion in polarized epithelial cells.

Increased plasma calcitonin levels in young spontaneously hypertensive rats: role in disturbed phosphate homeostasis.

In young spontaneously hypertensive rats (SHR) and in normotensive Wistar-Kyoto controls (WKY) several parameters of phosphate and calcium homeostasis were determined. At 6 and 8 weeks, blood analysis revealed a significant hypophosphatemia (p<0.001) in SHR and twice as high plasma calcitonin levels in SHR than in WKY controls. At 8 weeks, 1,25-dihydroxycholecalciferol concentration was 20% higher in SHR (p<0.02) while 25-hydroxycholecalciferol was unaltered (p<0.51). In addition total immunoreactive PTH, iPTH, was slightly increased (p<0.07) but intact PTH (1–84) (p>0.90) was not significantly different from age matched WKY controls. Also at 8 weeks, a slightly reduced serum ionized Ca2+ concentration (p<0.001) with no change in total serum calcium was found in SHR (p>0.39). Balance studies at 6 and 8 weeks of age revealed no significantly different balances for phosphate (F=2.5,p>0.10) and for calcium (F=2.6,p>0.09), although a tendency for slightly more positive balaces existed in SHR when compared to WKY. However, SHR excreted significantly less phosphate in the urine than WKY control (F=0.2,p<0.0009). Bone analysis was performed on femora of SHR and WKY of 6 weeks of age. Femora were significantly shorter in SHR (20.54±0.35 vs. 21.50±0.05 mm in WKY), whereas bone dry weight (127±6 vs. 107±2mg), bone ash weight (79±4 vs. 66±1 mg) and bone volume (0.196±0.007 vs. 0.165±0.004 cm3) were significantly greater in SHR. Calcium content per femur (717±35 vs. 617±11 μmol Ca/femur) and phosphate content per femur (512±23 vs. 447±8 μmol P/femur) were also significantly higher in SHR. It is discussed that the disturbances in phosphate homeostasis may be secondary to the strikingly increased plasma calcitonin levels present in young SHR.

A high phosphate diet lowers blood pressure in spontaneously hypertensive rats.

Plasma phosphate values are significantly lower in spontaneously hypertensive rats (SHR) than in normotensive Wistar-Kyoto rats (WKY). In this study, we increased plasma phosphate in SHR by a dietary phosphate intake and followed the effects on blood pressure. Fifteen male WKY and 15 male SHR were housed from 4 weeks of age up to 26 weeks. At 4 weeks of age all SHR manifested a hypophosphatemia compared with age-matched WKY (F = 62, p less than 0.0003). At 5 weeks of age, the rats were divided into three diet groups: a control group, a group receiving 1.41% (wt/vol) KCl in drinking water, and a group receiving 2% (wt/vol) K2HPO4 X KH2PO4 in drinking water. In the control (F = 16.2, p less than 0.02) and KCl groups, (F = 36.3, p less than 0.03), hypophosphatemia persisted throughout the study. The phosphate-supplemented diet normalized plasma phosphate level in SHR but did not change plasma phosphate level in WKY. As a consequence, no difference in plasma phosphate level between WKY and SHR was present in the group receiving additional phosphate from that time on (F = 1.2, p greater than 0.41). The phosphate-supplemented diet significantly decreased systolic blood pressure in both strains. In phosphate-supplemented SHR, a significant decline in systolic blood pressure was observed from 20 weeks of age on (at 20 weeks of age: 222 +/- 3 mm Hg for control SHR vs 198 +/- 5 mm Hg for phosphate-supplemented SHR; p less than 0.0003).(ABSTRACT TRUNCATED AT 250 WORDS)