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Proton pump inhibitors (PPIs) are potent blockers of gastric acid secretion, used by millions of patients suffering from gastric acid-related complaints. Although PPIs have an excellent safety profile, an increasing number of case reports describe patients with severe hypomagnesemia due to long-term PPI use. As there is no evidence of a renal Mg 2+ leak, PPI-induced hypomagnesemia is hypothesized to result from intestinal malabsorption of Mg 2+. The aim of this study was to investigate the effect of PPIs on Mg 2+ homeostasis in an in vivo mouse model. To this end, C57BL/6J mice were treated with omeprazole, under normal and low dietary Mg 2+ availability. Omeprazole did not induce changes in serum Mg 2+ levels (1.48 ± 0.05 and 1.54 ± 0.05 mmol/L in omeprazole-treated and control mice, respectively), urinary Mg 2+ excretion (35 ± 3 μmol/24 h and 30 ± 4 μmol/24 h in omeprazole-treated and control mice, respectively), or fecal Mg 2+ excretion (84 ± 4 μmol/24 h and 76 ± 4 μmol/24 h in omeprazole-treated and control mice, respectively) under any of the tested experimental conditions. However, omeprazole treatment did increase the mRNA expression level of the transient receptor potential melastatin 6 (TRPM6), the predominant intestinal Mg 2+ channel, in the colon (167 ± 15 and 100 ± 7 % in omeprazole-treated and control mice, respectively, P < 0.05). In addition, the expression of the colonic H +,K +-ATPase (cHK-α), a homolog of the gastric H +,K +-ATPase that is the primary target of omeprazole, was also significantly increased (354 ± 43 and 100 ± 24 % in omeprazole-treated and control mice, respectively, P < 0.05). The expression levels of other magnesiotropic genes remained unchanged. Based on these findings, we hypothesize that omeprazole inhibits cHK-α activity, resulting in reduced extrusion of protons into the large intestine. Since TRPM6-mediated Mg 2+ absorption is stimulated by extracellular protons, this would diminish the rate of intestinal Mg 2+ absorption. The increase of TRPM6 expression in the colon may compensate for the reduced TRPM6 currents, thereby normalizing intestinal Mg 2+ absorption during omeprazole treatment in C57BL/6J mice, explaining unchanged serum, urine, and fecal Mg 2+ levels. [ABSTRACT FROM AUTHOR]

The molecular assembly of the epithelial Ca2+ channels (TRPV5 and TRPV6) was investigated to determine the subunit stoichiometry and composition. Immunoblot analysis of Xenopus laevis oocytes expressing TRPV5 and TRPV6 revealed two specific bands of 75 and 85-100 kDa, corresponding to the core and glycosylated proteins, respectively, for each channel. Subsequently, membranes of these oocytes were sedimented on sucrose gradients. Immunoblotting revealed that TRPV5 and TRPV6 complexes migrate with a mol. wt of 400 kDa, in line with a tetrameric structure. The tetrameric stoichiometry was confirmed in an electrophysiological analysis of HEK293 cells co-expressing concatemeric channels together with a TRPV5 pore mutant that reduced Cd2+ sensitivity and voltage-dependent gating. Immunoprecipitations using membrane fractions from oocytes co-expressing TRPV5 and TRPV6 demonstrated that both channels can form heteromeric complexes. Expression of all possible heterotetrameric TRPV5/6 complexes in HEK293 cells resulted in Ca2+ channels that varied with respect to Ca2+-dependent inactivation, Ba2+ selectivity and pharmacological block. Thus, Ca2+-transporting epithelia co-expressing TRPV5 and TRPV6 can generate a pleiotropic set of functional heterotetrameric channels with different Ca2+ transport kinetics. [ABSTRACT FROM AUTHOR]

Rabbit pancreatic acinar cells, permeabilized by saponin treatment, rapidly accumulated 3.5 nmol of Ca2+/mg protein in an energy-dependent pool when incubated at an ambient free Ca2+ concentration of 100 nM. Maximal loading of the internal stores was reached at 10 min and remained unchanged thereafter. Complete inhibition of the Ca2+ pump with thapsigargin revealed that this plateau was the result of a steady-state between slow Ca2+ efflux and ATP-driven Ca2+ uptake. Sixty percent of the pool could be released by Ins(1,4,5)P3, whereas GTP released another twenty percent. The striking finding of this study is that the energy-dependent store could also be released by ruthenium red. Uptake experiments in the presence of ruthenium red revealed that the dye, at concentrations below 100 microM, selectively reduced the size of the Ins(1,4,5)P3-releasable pool. Ruthenium red had no effect on the half-maximal stimulatory concentration of Ins(1,4,5)P3. At concentrations beyond 100 microM, the dye also affected the GTP-releasable pool. Comparison with thapsigargin revealed that ruthenium red released Ca2+ from stores loaded to steady-state at a rate markedly faster than can be explained by inhibition of the ATPase alone. From the data presented, we concluded that ruthenium red selectively releases Ca2+ from the Ins(1,4,5)P3-sensitive store by activating a Ca2+ release channel, whereas Ca2+ release from the GTP-sensitive store is predominantly caused by inhibition of the Ca2+ pump. The postulated ruthenium red-sensitive Ca2+ release channel might be similar to the ryanodine-receptor in muscle. [ABSTRACT FROM AUTHOR]

Ca2+ homeostasis is an important factor, which is underlined by the numerous clinical symptoms that involve Ca2+ deficiencies. The overall Ca2+ balance is maintained by the concerted action of Ca2+ absorption in the intestine, reabsorption in the kidney, and exchange from bone, which are all under the control of the calciotropic hormones that are released upon a demand for Ca2+. In the kidney, these calciotropic hormones affect active Ca2+ reabsorption, which consists of TRPV5 as the apical entry gate for Ca2+ influx, calbindin-D28K as an intracellular ferry for Ca2+ and, NCX1 and PMCA1b for extrusion of Ca2+ across the basolateral membrane. This review highlights the action of hormones on renal Ca2+ handling and focuses on the coordinated control of the renal Ca2+ transport proteins. Parathyroid hormone stimulates renal Ca2+ handling by regulating active Ca2+ reabsorption on both the genomic and non-genomic level. Estrogens harbor calciotropic hormone characteristics positively regulating the expression of TRPV5, independently of vitamin D. Besides having a strong regulatory effect on the expression of the intestinal Ca2+ transport proteins, vitamin D contributes to the overall Ca2+ balance by enhancing the expression of the Ca2+ transport machinery in the kidney. Dietary Ca2+ is involved in regulating its own handling by controlling the expression of the renal Ca2+ transport proteins. Thus, the magnitude of Ca2+ entry via TRPV5 controls the expression of the other Ca2+ transport proteins underlining the gatekeeper function of this Ca2+ channel in the renal Ca2+ handling.Kidney International (2006) 69, 650–654. doi:10.1038/sj.ki.5000169; published online 11 January 2006 [ABSTRACT FROM AUTHOR]

The rising prevalence of kidney diseases urges the need for novel therapies. Kidney organoids and tubuloids are advanced in vitro models and have recently been described as promising tools to study kidney (patho)physiology. Recent developments have shown their application in disease modeling, drug screening, and nephrotoxicity. These applications rely on their ability to mimic (dys)function in vitro including endocrine activity and drug, electrolyte, and water transport. This review provides an overview of these emerging kidney models and focuses on the most recent developments that utilize their functional capabilities. In addition, we cover current limitations and provide future perspectives for this rapidly evolving field, including what these functional properties mean for translational and personalized medicine now and in the future., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Hypercalciuria is the most common metabolic risk factor in people with kidney stone disease. Its etiology is mostly multifactorial, although monogenetic causes of hypercalciuria have also been described. Despite the increased availability of genetic diagnostic tests, the vast majority of individuals with familial hypercalciuria remain unsolved. In this study, we investigated a consanguineous pedigree with idiopathic hypercalciuria. The proband additionally exhibited severe skeletal deformities and hyperparathyroidism. Whole-exome sequencing of the proband revealed a homozygous ultra-rare variant in TRPV5 (NM&#95;019841.7:c.1792G>A; p.(Val598Met)), which encodes for a renal Ca 2+ -selective ion channel. The variant segregates with the three individuals with hypercalciuria. The skeletal phenotype unique to the proband was due to an additional pathogenic somatic mutation in GNAS (NM&#95;000516.7:c.601C>T; p.(Arg201Cys)), which leads to polyostotic fibrous dysplasia. The variant in TRPV5 is located in the TRP helix, a characteristic amphipathic helix that is indispensable for the gating movements of TRP channels. Biochemical characterization of the TRPV5 p.(Val598Met) channel revealed a complete loss of Ca 2+ transport capability. This defect is caused by reduced expression of the mutant channel, due to misfolding and preferential targeting to the proteasome for degradation. Based on these findings, we conclude that biallelic loss of TRPV5 function causes a novel form of monogenic autosomal recessive hypercalciuria, which we name renal Ca 2+ -wasting hypercalciuria (RCWH). The recessive inheritance pattern explains the rarity of RCWH and underscores the potential prevalence of RCWH in highly consanguineous populations, emphasizing the importance of exploration of this disorder within such communities., (© 2024. The Author(s).)

Background: Disturbances in magnesium homeostasis are common in patients with chronic kidney disease (CKD) and are associated with increased mortality. The kidney is a key organ in maintaining normal serum magnesium concentrations. To this end, fractional excretion of magnesium (FEMg) increases as renal function declines. Despite recent progress, the hormonal regulation of renal magnesium handling is incompletely understood. Fibroblast Growth Factor 23 (FGF23) is a phosphaturic hormone that has been linked to renal magnesium handling. However, it has not yet been reported whether FGF23 is associated with renal magnesium handling in CKD patients., Methods: The associations between plasma FGF23 levels, plasma and urine magnesium concentrations and FEMg was investigated in a cross-sectional cohort of 198 non-dialysis CKD patients undergoing renal biopsy., Results: FGF23 was significantly correlated with FEMg (Pearson's correlation coefficient = 0.37, p<0.001) and urinary magnesium (-0.14, p=0.04), but not with plasma magnesium. The association between FGF23 and FEMg remained significant after adjusting for potential confounders, including estimated glomerular filtration rate (eGFR), parathyroid hormone and 25-hydroxyvitamin D., Conclusions: We report that plasma FGF23 is independently associated with measures of renal magnesium handling in a cohort of non-dialysis CKD patients. A potential causal relationship should be investigated in future studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Grigore, Zuidscherwoude, Witasp, Barany, Wernerson, Bruchfeld, Xu, Olauson and Hoenderop.)

Calcineurin inhibitors (CNIs) are immunosuppressive drugs used to prevent graft rejection after organ transplant. Common side effects include renal magnesium wasting and hypomagnesemia, which may contribute to new-onset diabetes mellitus, and hypercalciuria, which may contribute to post-transplant osteoporosis. Previous work suggested that CNIs reduce the abundance of key divalent cation transport proteins, expressed along the distal convoluted tubule, causing renal magnesium and calcium wasting. It has not been clear, however, whether these effects are specific for the distal convoluted tubule, and whether these represent off-target toxic drug effects, or result from inhibition of calcineurin. The CNI tacrolimus can inhibit calcineurin only when it binds with the immunophilin, FKBP12; we previously generated mice in which FKBP12 could be deleted along the nephron, to test whether calcineurin inhibition is involved, these mice are normal at baseline. Here, we confirmed that tacrolimus-treated control mice developed hypomagnesemia and urinary calcium wasting, with decreased protein and mRNA abundance of key magnesium and calcium transport proteins (NCX-1 and Calbindin-D 28k ). However, qPCR also showed decreased mRNA expression of NCX-1 and Calbindin-D 28k , and TRPM6. In contrast, KS-FKBP12 -/- mice treated with tacrolimus were completely protected from these effects. These results indicate that tacrolimus affects calcium and magnesium transport along the distal convoluted tubule and strongly suggests that inhibition of the phosphatase, calcineurin, is directly involved., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We explored the role of transient receptor potential vanilloid 4 (TRPV4) in murine bone metabolism and association of TRPV4 gene variants with fractures in humans. Urinary and histomorphometrical analyses demonstrated reduced osteoclast activity and numbers in male Trpv4(-/-) mice, which was confirmed in bone marrow-derived osteoclast cultures. Osteoblasts and bone formation as shown by serum procollagen type 1 amino-terminal propeptide and histomorphometry, including osteoid surface, osteoblast and osteocyte numbers were not affected in vivo. Nevertheless, osteoblast differentiation was enhanced in Trpv4(-/-) bone marrow cultures. Cortical and trabecular bone mass was 20% increased in male Trpv4(-/-) mice, compared to sex-matched wild type (Trpv4(+/+)) mice. However, at the same time intracortical porosity was increased and bone matrix mineralization was reduced. Together, these lead to a maximum load, stiffness and work to failure of the femoral bone, which were not different compared to Trpv4(+/+) mice, while the bone material was less resistant to stress and less elastic. The differential impacts on these determinants of bone strength were likely responsible for the lack of any changes in whole bone strength in the Trpv4(-/-) mice. None of these skeletal parameters were affected in female Trpv4(-/-) mice. The T-allele of rs1861809 SNP in the TRPV4 locus was associated with a 30% increased risk (95% CI: 1.1-1.6; p=0.013) for non-vertebral fracture risk in men, but not in women, in the Rotterdam Study. Meta-analyses with the population-based LASA study confirmed the association with non-vertebral fractures in men. This was lost when the non-population-based studies Mr. OS and UFO were included. In conclusion, TRPV4 is a male-specific regulator of bone metabolism, a determinant of bone strength, and a potential risk predictor for fractures through regulation of bone matrix mineralization and intra-cortical porosity. This identifies TRPV4 as a unique sexually dimorphic therapeutic and/or diagnostic candidate for osteoporosis., (© 2013.)

Background: α-Klotho is a protein mainly produced in the kidney. Its circulating form has been suggested to link renal damage and distant tissue pathology. As three assays to measure α-Klotho became commercially available, we performed an evaluation of these commercially available Klotho assays., Methods: We studied within-run variation, between-run variation, matrix effects, linearity, and recovery of added recombinant human Klotho in the α-Klotho assays of IBL (IBL International GmbH, Hamburg, Germany), Cusabio (Cusabio Biotech, Wuhan, China) and USCN (USCN life Science, Inc., Wuhan, China) using both serum and ethylenediaminetetraacetic acid plasma., Results: Within run variation was 4, 13 and 32% for the IBL, Cusabio and USCN assay, respectively. Agreement between serum and EDTA plasma was good in the IBL assay, but poor in the USCN and Cusabio assays however improved after modifications in the Cusabio assay. Standardization and agreement between assays was poor., Conclusions: The commercially available methods for the measurement of α-Klotho differ in quality. Some of the manufacturers should improve their assays in order to produce accurate results so that reliable conclusions can be drawn from studies in which these assays are used.

During chronic kidney disease (CKD), drug metabolism is affected leading to changes in drug disposition. Furthermore, there is a progressive accumulation of uremic retention solutes due to impaired renal clearance. Here, we investigated whether uremic toxins can influence the metabolic functionality of human conditionally immortalized renal proximal tubule epithelial cells (ciPTEC) with the focus on UDP-glucuronosyltransferases (UGTs) and mitochondrial activity. Our results showed that ciPTEC express a wide variety of metabolic enzymes, including UGTs. These enzymes were functionally active as demonstrated by the glucuronidation of 7-hydroxycoumarin (7-OHC; K(m) of 12±2μM and a V(max) of 76±3pmol/min/mg) and p-cresol (K(m) of 33±13μM and a V(max) of 266±25pmol/min/mg). Furthermore, a wide variety of uremic toxins, including indole-3-acetic acid, indoxyl sulfate, phenylacetic acid and kynurenic acid, reduced 7-OHC glucuronidation with more than 30% as compared with controls (p<0.05), whereas UGT1A and UGT2B protein expressions remained unaltered. In addition, our results showed that several uremic toxins inhibited mitochondrial succinate dehydrogenase (i.e. complex II) activity with more than 20% as compared with controls (p<0.05). Moreover, indole-3-acetic acid decreased the reserve capacity of the electron transport system with 18% (p<0.03). In conclusion, this study shows that multiple uremic toxins inhibit UGT activity and mitochondrial activity in ciPTEC, thereby affecting the metabolic capacity of the kidney during CKD. This may have a significant impact on drug and uremic retention solute disposition in CKD patients., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Ca(2+) is an essential ion in all organisms and many physiological functions in the body rely on the exact maintenance of the Ca(2+) balance. The epithelial Ca(2+) channels TRPV5 [TRP (transient receptor potential) vanilloid 5] and TRPV6 are the most Ca(2+)-selective members of the TRP superfamily and are generally considered as the gatekeepers of Ca(2+) entry across epithelia. TRPV5 is involved in Ca(2+) reabsorption from pro-urine, while TRPV6 has an essential role in intestinal Ca(2+) uptake. These channels are the prime targets of calciotropic hormonal regulation, including vitamin D and parathyroid hormone. In addition, extra- and intra-cellular signalling by associated proteins and Ca(2+) itself play key roles in TRPV5 and TRPV6 regulation. In this paper, we describe the present understanding of the concerted action of calbindin-D(28k), klotho and BSPRY (B-box and SPRY-domain-containing protein) at different levels throughout the epithelial cell to control Ca(2+) influx at the luminal entry gate.

Blood calcium concentration is maintained within a narrow range despite large variations in dietary input and body demand. The Transient Receptor Potential ion channel TRPV5 has been implicated in this process. We report here that TRPV5 is stimulated by the mammalian hormone klotho. Klotho, a beta-glucuronidase, hydrolyzes extracellular sugar residues on TRPV5, entrapping the channel in the plasma membrane. This maintains durable calcium channel activity and membrane calcium permeability in kidney. Thus, klotho activates a cell surface channel by hydrolysis of its extracellular N-linked oligosaccharides.

The molecular assembly of the epithelial Ca(2+) channels (TRPV5 and TRPV6) was investigated to determine the subunit stoichiometry and composition. Immunoblot analysis of Xenopus laevis oocytes expressing TRPV5 and TRPV6 revealed two specific bands of 75 and 85-100 kDa, corresponding to the core and glycosylated proteins, respectively, for each channel. Subsequently, membranes of these oocytes were sedimented on sucrose gradients. Immuno blotting revealed that TRPV5 and TRPV6 complexes migrate with a mol. wt of 400 kDa, in line with a tetrameric structure. The tetrameric stoichiometry was confirmed in an electrophysiological analysis of HEK293 cells co-expressing concatemeric channels together with a TRPV5 pore mutant that reduced Cd(2+) sensitivity and voltage-dependent gating. Immuno precipitations using membrane fractions from oocytes co-expressing TRPV5 and TRPV6 demonstrated that both channels can form heteromeric complexes. Expression of all possible heterotetrameric TRPV5/6 complexes in HEK293 cells resulted in Ca(2+) channels that varied with respect to Ca(2+)-dependent inactivation, Ba(2+) selectivity and pharmacological block. Thus, Ca(2+)-transporting epithelia co-expressing TRPV5 and TRPV6 can generate a pleiotropic set of functional heterotetrameric channels with different Ca(2+) transport kinetics.

The calcium release-activated calcium channel (CRAC) is a highly Ca(2+)-selective ion channel that is activated on depletion of inositol triphosphate (IP(3))-sensitive intracellular Ca(2+) stores. It was recently reported that CaT1, a member of the TRP family of cation channels, exhibits the unique biophysical properties of CRAC, which led to the conclusion that CaT1 comprises all or part of the CRAC pore (Yue, L., Peng, J. B., Hediger, M. A., and Clapham, D. E. (2001) Nature 410, 705-709). Here, we directly compare endogenous CRAC with heterologously expressed CaT1 and show that they manifest several clearly distinct properties. CaT1 can be distinguished from CRAC in the following features: sensitivity to store-depleting agents; inward rectification in the absence of divalent cations; relative permeability to Na(+) and Cs(+); effect of 2-aminoethoxydiphenyl borate (2-APB). Moreover, CaT1 displays a mode of voltage-dependent gating that is fully absent in CRAC and originates from the voltage-dependent binding/unbinding of Mg(2+) inside the channel pore. Our results imply that the pores of CaT1 and CRAC are not identical and indicate that CaT1 is a Mg(2+)-gated channel not directly related to CRAC.

The organization of Na(+) and Ca(2+) transport pathways along the mouse distal nephron is incompletely known. We revealed by immunohistochemistry a set of Ca(2+) and Na(+) transport proteins along the mouse distal convolution. The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) characterized the distal convoluted tubule (DCT). The amiloride-sensitive epithelial Na(+) channel (ENaC) colocalized with NCC in late DCT (DCT2) and extended to the downstream connecting tubule (CNT) and collecting duct (CD). In early DCT (DCT1), the basolateral Ca(2+)-extruding proteins [Na(+)/Ca(2+) exchanger (NCX), plasma membrane Ca(2+)-ATPase (PCMA)] and the cytoplasmic Ca(2+)-binding protein calbindin D(28K) (CB) were found at very low levels, whereas the cytoplasmic Ca(2+)/Mg(2+)-binding protein parvalbumin was highly abundant. NCX, PMCA, and CB prevailed in DCT2 and CNT, where we located the apical epithelial Ca(2+) channel (ECaC1). Its subcellular localization changed from apical in DCT2 to exclusively cytoplasmic at the end of CNT. NCX and PMCA decreased in parallel with the fading of ECaC1 in the apical membrane. All three of them were undetectable in CD. These findings disclose DCT2 and CNT as major sites for transcellular Ca(2+) transport in the mouse distal nephron. Cellular colocalization of Ca(2+) and Na(+) transport pathways suggests their mutual interactions in transport regulation.

Rickets and hyperparathyroidism caused by a defective vitamin D receptor (VDR) can be prevented in humans and animals by high calcium intake, suggesting that intestinal calcium absorption is critical for 1,25(OH)(2) vitamin D [1,25(OH)(2)D(3)] action on calcium homeostasis. We assessed the rate of serum (45)Ca accumulation within 10 min of oral gavage in two strains of VDR-knockout (KO) mice (Leuven and Tokyo KO) and observed a 3-fold lower area under the curve in both KO strains. Moreover, we evaluated the expression of intestinal candidate genes involved in transcellular calcium transport. The calcium transport protein1 (CaT1) was more abundantly expressed at mRNA level than the epithelial calcium channel (ECaC) in duodenum, but both were considerably reduced (CaT1>90%, ECaC>60%) in the two VDR-KO strains on a normal calcium diet. Calbindin-D(9K) expression was decreased only in the Tokyo KO, whereas plasma membrane calcium ATPase (PMCA(1b)) expression was normal in both VDR-KOs. In Leuven wild-type mice, a high calcium diet inhibited (>90%) and 1,25(OH)(2)D(3) injection or low calcium diet induced (6-fold) duodenal CaT1 expression and, to a lesser degree, ECaC and calbindin-D(9K) expression. In Leuven KO mice, however, high or low calcium intake decreased calbindin-D(9K) and PMCA(1b) expression, whereas CaT1 and ECaC expression remained consistently low on any diet. These results suggest that the expression of the novel duodenal epithelial calcium channels (in particular CaT1) is strongly vitamin D-dependent, and that calcium influx, probably interacting with calbindin-D(9K), should be considered as a rate-limiting step in the process of vitamin D-dependent active calcium absorption.

1. The recent identification of the epithelial Ca(2+) channel, ECaC1, represents a major step forward in our knowledge of renal Ca(2+) handling. ECaC1 constitutes the rate-limiting apical Ca(2+) entry mechanism of active, transcellular Ca(2+) reabsorption. This unique highly selective Ca(2+) channel shares a low but significant homology with transient receptor potential (TRP) channels and vanilloid receptors (VR). 2. We have studied the pharmacological modulation of currents through ECaC1 heterologously expressed in HEK 293 cells. Monovalent cation currents were measured by use of the whole cell patch clamp technique in cells dialysed with 10 mM BAPTA or 10 mM EGTA to prevent the fast Ca(2+) dependent inactivation of ECaC1. 3. Several modulators were tested, including inorganic cations, putative store-operated Ca(2+) entry (SOC) blockers, the vanilloid receptor (VR-1) blocker capsazepine, protein tyrosine kinase blockers, calmodulin antagonists and ruthenium red. 4. Ruthenium red and econazole appeared to be the most effective inhibitors of currents through ECaC1, with IC(50) values of 111 nM and 1.3 microM, respectively, whereas the selective SOC inhibitor, SKF96365, was nearly ineffective. 5. The divalent cation current block profile for ECaC1 is Pb(2+)=Cu(2+) >Zn(2+) >Co(2+) >Fe(2+) with IC(50) values between 1 and approximately 10 microM. 6. In conclusion, ECaC activity is effectively inhibited by various compounds including ruthenium red, antimycotic drugs and divalent cations, which might be useful tools for pharmacological manipulation and several disorders related to Ca(2+) homeostasis could benefit from such developments.

We have studied the modulation by intracellular Ca2+ of the epithelial Ca2+ channel, ECaC, heterologously expressed in HEK 293 cells. Whole-cell and inside-out patch clamp current recordings were combined with FuraII-Ca2+ measurements:1. Currents through ECaC were dramatically inhibited if Ca2+ was the charge carrier. This inhibition was dependent on the extracellular Ca2+ concentration and occurred also in cells buffered intracellularly with 10 mM BAPTA.2. Application of 30 mM [Ca(2)]e induced in non-Ca2+] buffered HEK 293 cells at -80 m V an increase in intracellular Ca2+([Ca2]i) with a maximum rate of rise of 241 +/-15nM/s (n= 18 cells) and a peak value of 891 +/- 106 nM. The peak of the concomitant current with a density of 12.3 +/- 2.6 pA/pF was closely correlated with the peak of the first-time derivative of the Ca2+ transient, as expected if the Ca2+ transient is due to influx of Ca2+. Consequently, no Ca2+] signal was observed in cells transfected with the Ca2+ impermeable ECaC mutant, D542A, in which an aspartate in the pore region was neutralized.3. Increasing [Ca2+]i by dialyzing the cell with pipette solutions containing various Ca2+] concentrations, all buffered with 10 mM BAPTA, inhibited currents through ECaC carried by either Na+ or Ca2+] ions. Half maximal inhibition of Ca(2+)currents in the absence of monovalent cations occurred at 67 nM (n between 6 and 8), whereas Na+ currents in the absence of Ca2+] and Mg2+ were inhibited with an IC50 of 89 nM (n between 6 and 10). Currents through ECaC in the presence of 1 mM Ca2+ and Na+, which are mainly carried by Ca2+, are inhibited by [Ca2]i with an IC50of 82 nM (n between 6 and 8). Monovalent cation currents through the Ca2+impermeable D542A ECaC mutant were also inhibited by an elevation of [Ca2]i (IC50 = 123 nM, n between 7 and 18). 4. The sensitivity of ECaC currents in inside-out patches for [Ca2]i was slightly shifted to higher concentrations as compared with whole cell measurements. Half-maximal inhibition occurred at 169 nM if Na+ was the charge carrier (n between 4 and 11) and 228 nM at 1 mM [Ca2]e (n between 4 and 8).5. Recovery from inhibition upon washout of extracellular Ca2+ (whole-cell configuration) or removal of Ca2+ from the inner side of the channel (inside-out patches) was slow in both conditions. Half-maximal recovery was reached after 96 +/- 34 s (n= 15) in whole-cell mode and after 135 +/- 23 s (n = 17) in inside-out patches.6. We conclude that influx of Ca2+ through ECaC and [Ca2]i induce feedback inhibition of ECaC currents, which is controlled by the concentration of Ca2+ in a micro domain near the inner mouth of the channel. Slow recovery seems to depend on dissociation of Ca( 2+ from an internal Ca2+ binding site at ECaC., (Copyright 2001 Harcourt Publishers Ltd.)

We investigated the effect of extracellular pH on whole-cell currents through the epithelial Ca2+ channel, ECaC, expressed in HEK 293 cells. Both mono- and divalent current densities were significantly smaller at pH 6.0 than at pH 7.4. At pH 8.5 they were slightly larger. Lowering extracellular pH enhanced the slow component of monovalent current activation at negative potentials but had no significant effect on the kinetics of Ca2+ currents. The kinetics of block of monovalent cation current by extracellular Mg2+ was significantly changed at high and low pH. The time constant of the time- and voltage-dependent current component during a voltage step to -140 mV was significantly larger at pH 8.5 than at pH 7.4. At pH 6.0 it was almost absent. The [Mg2+] inhibiting 50% of monovalent current through ECaC at pH 6.0 (IC50) was 323 +/- 23 microM (n = 8), compared with 62 +/- 9 microM (n = 4) at pH 7.4 and 38 +/- 4 microM (n = 8) at pH 8.5. The affinity of ECaC for Ca2+ was also affected by extracellular pH, shifting from 4.8 +/- 0.7 microM (n = 6) at pH 6.0 to 161 +/- 30 nM (n = 5) at pH 7.4 and 425 +/- 117 nM (n = 8) at pH 8.5.

Phospholipase D (PLD) is distributed widely in mammalian tissues where it is believed to play an important role in the regulation of cell functions and cell fate by a variety of extracellular signals. In this study, we used primary cultures of rabbit connecting tubule (CNT) and cortical collecting duct (CCD) cells, grown to confluence on a permeable support, to investigate the possible involvement of PLD in the mechanism of action of hormones that regulate Ca(2+) reabsorption. RT-PCR revealed the presence of transcripts of PLD1b and PLD2, but not PLD1a, in these cultures. Moreover, the expression of substantial amounts of PLD1 protein was demonstrated by Western blotting. To measure PLD activity, cells were labelled with [(3)H]myristic acid after which the PLD-catalysed formation of radiolabelled phosphatidylethanol ([(3)H]PtdEth) was measured in the presence of 1% (v/v) ethanol. Deamino-Cys,D-Arg(8)-vasopressin (dDAVP) and N(6)-cyclopentyladenosine (CPA), two potent stimulators of Ca(2+) transport across these monolayers, stimulated PLD activity as was indicated by a marked increase in [(3)H]PtdEth. Similarly, ATP, a potent inhibitor of dDAVP- and CPA-stimulated Ca(2+) transport, increased the formation of [(3)H]PtdEth. PLD activity was furthermore increased by 8Br-cAMP and following acute (30 min) stimulation of protein kinase C (PKC) with a phorbol ester (PMA). Chronic PMA treatment (120 h) to downregulate phorbol ester-sensitive PKC isoforms did not affect PLD activation by dDAVP, CPA and 8Br-cAMP, while markedly decreasing the effect of ATP and abolishing the effect of PMA. The PKC inhibitor chelerythrine significantly reduced PLD activation by dDAVP, CPA and 8Br-cAMP, without changing the effect of ATP. The inhibitor only partially reduced the effect of PMA. This study shows that Ca(2+) transporting cells of CNT and CCD contain a regulated PLD activity. The physiological relevance of this activity, which is not involved in Ca(2+) reabsorption, remains to be established.

We have used the whole-cell patch-clamp technique to analyse the permeation properties and ionic block of the epithelial Ca2+ channel ECaC heterologously expressed in human embryonic kidney (HEK) 293 cells. Cells dialysed with 10 mM BAPTA and exposed to Ca2+-containing, monovalent cation-free solutions displayed large inwardly rectifying currents. Their reversal potential depended on the extracellular Ca2+ concentration, [Ca2+]o. The slope of the relationship between reversal potential and [Ca2+]o on a logarithmic scale was 21 +/- 4 mV, compared with 29 mV as predicted by the Nernst equation (n = 3-5 cells). Currents in mixtures of Ca2+ and Na+ or Ca2+ and Ba2+ showed anomalous mole fraction behaviour. We have described the current-concentration plot for Ca2+ and Na+ by a kinetic permeation model, i.e. the "step" model. Extracellular Mg2+ blocked both divalent and monovalent currents with an IC50 of 62 +/- 9 microM(n = 4) in Ca2+-free conditions and 328 +/- 50 microM (n = 4-9) in 100 microM Ca2+ solutions. Mono- and divalent currents through ECaCs were blocked by gadolinium, lanthanum and cadmium, with a blocking order of Cd2+ >> Gd3+ > La3+. We conclude that the permeation of monovalent and divalent cations through ECaCs shows similarities with L-type voltage-gated Ca2+ channels, the main differences being a higher Ca2+ affinity and a significantly higher current density in micromolar Ca2+ concentrations in the case of ECaCs.

The epithelial Ca(2+) channel (ECaC), which was recently cloned from rabbit kidney, exhibits distinctive properties that support a facilitating role in transcellular Ca(2+) (re)absorption. ECaC is structurally related to the family of six transmembrane-spanning ion channels with a pore-forming region between S5 and S6. Using point mutants of the conserved negatively charged amino acids present in the putative pore, we have identified a single aspartate residue that determines Ca(2+) permeation of ECaC and modulation by extracellular Mg(2+). Mutation of the aspartate residue, D542A, abolishes Ca(2+) permeation and Ca(2+)-dependent current decay as well as block by extracellular Mg(2+), whereas monovalent cations still permeate the mutant channel. Variation of the side chain length in mutations D542N, D542E, and D542M attenuated Ca(2+) permeability and Ca(2+)-dependent current decay. Block of monovalent currents through ECaC by Mg(2+) was decreased. Exchanging the aspartate residue for a positively charged amino acid, D542K, resulted in a nonfunctional channel. Mutations of two neighboring negatively charged residues, i.e. Glu(535) and Asp(550), had only minor effects on Ca(2+) permeation properties.