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Ultrastructural and Immunohistochemical Localization of the Plasma Membrane Ca 2+ -ATPase 4 (PMCA4) in Ca 2+ -Transporting Epithelia
Abstract
Plasma Membrane Ca(2+)-ATPase's (PMCA) participate in epithelial Ca(2+) transport and intracellular Ca(2+) signaling. The Pmca4 isoform is enriched in distal nephron isolates and decreased in mice lacking the epithelial Ca(2+) channel, Trpv5. We therefore hypothesized that Pmca4 plays a significant role in transcellular Ca(2+) flux and investigated the localization and regulation of Pmca4 in Ca(2+)-transporting epithelia. Using antibodies directed specifically against Pmca4, we found it expressed only in the smooth muscle layer of mouse and human intestine, while pan-specific Pmca antibodies detected Pmca1 in lateral membranes of enterocytes. In kidney, Pmca4 showed broad localization to the distal nephron. In mouse, expression was most abundant in segments coexpressing the epithelial Ca(2+) channel, Trpv5. Significant, albeit lower expression, was also evident in the region encompassing the cortical thick ascending limbs, macula densa, and early distal tubules as well as smooth muscle layers surrounding renal vessels. In human kidney, a similar pattern of distribution was observed, with highest PMCA4 expression in NCC positive tubules. Electron microscopy demonstrated Pmca4 localization in distal nephron cells at both the basolateral membrane and intracellular perinuclear compartments, but not submembranous vesicles, suggesting rapid trafficking to the plasma membrane is unlikely to occur in vivo. Pmca4 expression was not altered by perturbations in Ca(2+) balance, pointing to a housekeeping function of the pump in Ca(2+) transporting epithelia. In conclusion, Pmca4 shows a divergent expression pattern in Ca(2+) transporting epithelia, inferring diverse roles for this isoform not limited to transepithelial Ca(2+) transport.
Copyright © 2014, American Journal of Physiology - Renal Physiology.
... In the current model, Ca 2ϩ enters the epithelial cell via the apical Ca 2ϩ channel, transient receptor potential vanilloid 6 (TRPV6) (4,62), from where it is shuttled across the cell bound to calbindin-D 9K (CaBP 9K ) (24). Basolateral extrusion is mediated by Ca 2ϩ -ATPase (PMCA1) and, to a lesser extent, the Na ϩ -Ca 2ϩ exchanger (NCX1) (1,24,55). Similar to Ca 2ϩ , Mg 2ϩ is absorbed in the intestine via transcellular and paracellular pathways (3). Absorption in the small intestine is thought to occur predominantly via the paracellular route (3,40). ...
... This structure encompasses the distal convoluted tubule (DCT segments 1 and 2, the connecting tubule (CNT), and initial cortical collecting duct (38). Akin to the duodenum, Ca 2ϩ transport in the distal convolution (primarily, DCT2 and CNT) involves the apical Ca 2ϩ channel TRPV5, intracellular binding, and shuttling by calbindin-D 28K (CaBP28K) and basolateral extrusion via Ca 2ϩ -ATPases (PMCA1 and PMCA4) or NCX1 (1,16,23,24). Vectorial transcellular Mg 2ϩ transport occurs in the DCT1 and is facilitated by apical uptake of Mg 2ϩ via TRPM6 channels (20). ...
... Isolation of intestinal segments, kidney, and mammary fat pads. Isolation of murine tissue was performed as previously described (1), and the tissue was saved for biochemical and histological analysis. The first 4.6 cm of intestine distal to the pylorus was considered to be the duodenum. ...
Significant alterations in maternal calcium (Ca2+) and magnesium (Mg2+) balance occur during lactation. Ca2+ is the primary divalent cation mobilized into breast milk by demineralization of the skeleton and alterations in intestinal and renal Ca2+ transport. Mg2+ is also concentrated in breast milk, but the underlying mechanisms are not well understood. To determine the molecular alterations in Ca2+ and Mg2+ transport in the intestine and kidney during lactation, 3 groups of female mice consisting of either non-pregnant controls, lactating mice, or mice undergoing involution were examined. The fractional excretion of Ca2+, but not Mg2+, rose significantly during lactation. Renal 1-alpha hydroxylase and 24-OHase mRNA levels increased markedly as did plasma 1,25 dihydroxyvitamin D levels. This was accompanied by significant increases in intestinal expression of Trpv6 and S100g in lactating mice. However, no alterations in the expression of cation permeable claudins (-2, -12 or -15) were found in the intestine. In kidney, increased expression of Trpv5 and Calb1 was observed during lactation, while no changes in claudins involved in Ca2+ and Mg2+ transport (-2, -14, -16 or -19) were found. Consistent with the mRNA expression, both Calbindin-D28K and TRPV5 protein expression increased. Colonic Trpm6 expression increased during lactation, while renal Trpm6 remained unaltered. In conclusion, proteins involved in transcellular Ca2+ and Mg2+ transport pathways increase during lactation, while expression of paracellular transport proteins remained unchanged. Increased fractional Ca2+ excretion can be explained by vitamin D-dependent intestinal hyperabsorption and bone demineralization, despite enhanced transcellular Ca2+ uptake by the kidney.
... 25 In vivo calcium absorption studies in 12-week-old CaBP9K knockout mice showed no difference to wildtype mice, suggesting that CaBP9K is not essential for intestinal calcium absorption. 35 The basolateral extrusion of calcium from the enterocyte is mediated by plasma membrane Ca 2þ -ATPase (PMCA1) and the Na þ /Ca 2þ exchanger (NCX1) 1. 1,36 In humans, PMCA1 is expressed in the duodenum, ileum, and colon, whereas in the mouse Pmca1 is expressed along all segments of the small and large intestine. 24 In the duodenum of rabbits, PMCA colocalizes with TRPV6 and CaBP9K. ...
... 78 In human and mouse kidney, PMCA4 is expressed in the distal nephron, and in mice it is most highly expressed in cells with TRPV5. 36 This suggests a role in calcium reabsorption, although its expression is not altered by calcium intake and Pmca4 knockout mice do not display an altered calcium phenotype, thereby inferring a housekeeping role. 36,79 An Ncx1 knockout model is embryonically lethal due to its importance in cardiomyocyte development. ...
... 36 This suggests a role in calcium reabsorption, although its expression is not altered by calcium intake and Pmca4 knockout mice do not display an altered calcium phenotype, thereby inferring a housekeeping role. 36,79 An Ncx1 knockout model is embryonically lethal due to its importance in cardiomyocyte development. However, work in cell culture models support the idea that NCX1 significantly contributes to basolateral calcium extrusion in the distal nephron. ...
Calcium is vital for many physiological functions including bone mineralization. Postnatal deposition of calcium into bone is greatest in infancy and continues through childhood and adolescence until peek mineral density is reached in early adulthood. Thereafter, bone mineral density remains static until it eventually declines in later life. A positive calcium balance, i.e. more calcium absorbed than excreted, is crucial to bone deposition during growth and thus to peek bone mineral density. Dietary calcium is absorbed from the intestine into the blood. It is then filtered by the renal glomerulus and either reabsorbed by the tubule or excreted in the urine. Calcium can be (re)absorbed across intestinal and renal epithelia via both transcellular and paracellular pathways. Current evidence suggests that significant intestinal and renal calcium transport changes occur throughout development. However, the molecular details of these alterations are incompletely delineated. Here we first briefly review the current model of calcium transport in the intestine and renal tubule in the adult. Then, we describe what is known with regard to calcium handling through postnatal development, and how alterations may aid in mediating a positive calcium balance. The role of transcellular and paracellular calcium transport pathways and the contribution of specific intestinal and tubular segments vary with age. However, the current literature highlights knowledge gaps in how specifically intestinal and renal calcium (re)absorption occurs early in postnatal development. Future research should clarify the specific changes in calcium transport throughout early postnatal development including mediators of these alterations enabling appropriate bone mineralization.
Impact statement
This mini review outlines the current state of knowledge pertaining to the molecules and mechanisms maintaining a positive calcium balance throughout postnatal development. This process is essential to achieving optimal bone mineral density in early adulthood, thereby lowering the lifetime risk of osteoporosis.
... Species differences are observed in the arrangement of the above-mentioned segments within the distal convolution. As such, the subsegmentation of the DCT as well as the histolog- (84,153,195,223,247). PMCA4 and NCX1 are highly abundant in the DCT2 and CNT region of the mouse, colocalizing with TRPV5; however, weaker expression of the basolateral Ca 2ϩ extrusion protein have also been noted in DCT (not displayed in figure) (8,149). Although the MR is expressed in the DCT, no effect on NCC has been documented, when the MR is deleted (48). ...
... In mouse, TRPV5 localizes with calbindin-D 28K , which may function as an intracellular Ca 2ϩ buffer and aid in shuttling Ca 2ϩ from apical to basolateral aspects of the cell (131). Extrusion of Ca 2ϩ from the cell into renal interstitium is driven by the Na 2ϩ /Ca 2ϩ exchanger type 1 (NCX1) and the plasma membrane Ca 2ϩ -ATPase proteins (PMCA1 and PMCA4) (8,197,239). Both NCX and PMCA4 are highly abundant in the DCT2 and CNT region of the mouse, colocalizing with TRPV5; however, weaker expression of the basolateral Ca 2ϩ extrusion protein have also been noted in DCT (not depicted in Fig. 3), although no apical Ca 2ϩ channel has been documented in the segment (8,149). ...
... Extrusion of Ca 2ϩ from the cell into renal interstitium is driven by the Na 2ϩ /Ca 2ϩ exchanger type 1 (NCX1) and the plasma membrane Ca 2ϩ -ATPase proteins (PMCA1 and PMCA4) (8,197,239). Both NCX and PMCA4 are highly abundant in the DCT2 and CNT region of the mouse, colocalizing with TRPV5; however, weaker expression of the basolateral Ca 2ϩ extrusion protein have also been noted in DCT (not depicted in Fig. 3), although no apical Ca 2ϩ channel has been documented in the segment (8,149). Targeted transgenic strategies have been implemented to understand the contribution of these genes to overall Ca 2ϩ transport within the distal convolution. ...
Calcium (Ca2+) and Magnesium (Mg2+) reabsorption along the renal tubule is dependent on distinct trans- and paracellular pathways. Our understanding of the molecular machinery involved is increasing. Ca2+ and Mg2+ reclamation in kidney is dependent on a diverse array of proteins, which are important for both forming divalent cation permeable pores and channels, but also for generating the necessary driving forces for Ca2+ and Mg2+ transport. Alterations in these molecular constituents lead to profound effects on tubular Ca2+ and Mg2+ handling. Diuretics are used to treat a large range of clinical conditions, but most commonly for the management of blood pressure and fluid balance. The pharmacological targets of diuretics generally directly facilitate sodium (Na+) transport, but also indirectly affect renal Ca2+ and Mg2+ handling, i.e. by establishing a prerequisite electrochemical gradient. It is therefore not surprising that substantial alterations in divalent cation handling can be observed following diuretic treatment. The effects of diuretics on renal Ca2+ and Mg2+ handling are reviewed in the context of the current understanding of basal molecular mechanisms of Ca2+ and Mg2+ transport. Acetazolamide, osmotic diuretics, NHE3 inhibitors and antidiabetic SGLT blocking compounds, target the proximal tubule, where paracellular Ca2+ transport predominates. Loop-diuretics and ROMK inhibitors block thick ascending limb transport, a segment with significant paracellular Ca2+ and Mg2+ transport. Thiazides target the distal convoluted tubule, however, their effect on divalent cation transport is not limited to that segment. Finally, potassium-sparing diuretics, which inhibit electrogenic Na+ transport at distal sites, can also affect divalent cation transport.
... PMCA1 is considered as the predominant PMCA responsible for transcellular Ca 2+ transport in the late DCT and CNT [18,19]. Recently however, Alexander et al. investigated the exact localization of PMCA4 in the kidney, by co-staining the different tubular segments with representative markers [20]. They verified that PMCA4 was expressed highest in tubules that also expressed TRPV5. ...
... Importantly, for the first time, this study aimed to characterize the renal role of PMCA4. PMCA4 has recently been localized in Ca 2+ transporting epithelial cells, promoting speculation about its functional role in transcellular Ca 2+ transport at the distal nephron [20,22]. In this study we show that ablation of PMCA4 has no significant effect on serum Ca 2+ level or on renal Ca 2+ excretion. ...
... However, Van der Hagen et al. showed that there was relatively more mRNA expression of PMCA4 in the late DCT and CNT compared to total kidney, whereas PMCA1 was not enriched in this segment [22]. This was confirmed at the protein level, where the most intense expression of PMCA4 in the kidney was observed in TRPV5 expressing tubules [20,22]. In addition, the TRPV5 KO mouse showed a decrease in mRNA levels of CaBP 28k, NCX1 and PMCA4, but not PMCA1 [22]. ...
Calcium (Ca2+) is vital for multiple processes in the body, and maintenance of the electrolyte concentration is required for everyday physiological function. In the kidney, and more specifically, in the late distal convoluted tubule and connecting tubule, the fine-tuning of Ca2+ reabsorption from the pro-urine takes place. Here, Ca2+ enters the epithelial cell via the transient receptor potential vanilloid receptor type 5 (TRPV5) channel, diffuses to the basolateral side bound to calbindin-D28k and is extruded to the blood compartment via the Na+/Ca2+ exchanger 1 (NCX1) and the plasma membrane Ca2+ ATPase (PMCA). Traditionally, PMCA1 was considered to be the primary Ca2+ pump in this process. However, in recent studies TRPV5-expressing tubules were shown to highly express PMCA4. Therefore, PMCA4 may have a predominant role in renal Ca2+ handling. This study aimed to elucidate the role of PMCA4 in Ca2+ homeostasis by characterizing the Ca2+ balance, and renal and duodenal Ca2+-related gene expression in PMCA4 knockout mice. The daily water intake of PMCA4 knockout mice was significantly lower compared to wild type littermates. There was no significant difference in serum Ca2+ level or urinary Ca2+ excretion between groups. In addition, renal and duodenal mRNA expression levels of Ca2+-related genes, including TRPV5, TRPV6, calbindin-D28k, calbindin-D9k, NCX1 and PMCA1 were similar in wild type and knockout mice. Serum FGF23 levels were significantly increased in PMCA4 knockout mice. In conclusion, PMCA4 has no discernible role in normal renal Ca2+ handling as no urinary Ca2+ wasting was observed. Further investigation of the exact role of PMCA4 in the distal convoluted tubule and connecting tubule is required.
... Mice were then euthanized by cervical dislocation and kidneys were isolated as previously. 26 Data are shown in Figure 1. ...
... RNA was extracted from the tissues using TRIzol (Invitrogen, Carlsbad, CA, USA) and processed as previously. 26,29 In brief, after DNAse treatment (Invitrogen, Carlsbad, CA, USA), cDNA synthesis was performed using the SensiFAST cDNA Synthesis Kit (Bioline, London, UK). Then, qPCR was performed in triplicate for each sample on the QuantStudio 6 Pro Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) using TaqMan Master Mix (Applied Biosystems, Waltham, MA, USA) and specific probes and primers (Table S1). ...
Background:
Parathyroid Ca2+-sensing receptor (CaSR) activation inhibits parathyroid hormone (PTH) release, while activation of renal CaSRs attenuates Ca2+ transport and increases expression of the pore-blocking claudin-14. Patients with autosomal dominant hypocalcemia 1 (ADH1), due to activating CASR mutations, exhibit hypocalcemia but not always hypercalciuria (elevated Ca2+ in urine). The latter promotes nephrocalcinosis and renal insufficiency. Although CaSRs throughout the body including the kidney harbor activating CASR mutations, it is not understood why only some ADH1 patients display hypercalciuria.
Methods:
Activation of the CaSR was studied in mouse models and a ADH1 patient. In vitro CaSR activation was studied in HEK293 cells.
Findings:
Cldn14 showed blood Ca2+ concentration-dependent regulation, which was absent in mice with kidney-specific Casr deletion, indicating Cldn14 is a suitable marker for chronic CaSR activation in the kidney. Mice with a gain-of-function mutation in the Casr (Nuf) were hypocalcemic with low plasma PTH levels. However, renal CaSRs were not activated at baseline but only after normalizing blood Ca2+ levels. Similarly, significant hypercalciuria was not observed in a ADH1 patient until blood Ca2+ was normalized. In vitro experiments indicate that increased CaSR expression in the parathyroid relative to the kidney could contribute to tissue-specific CaSR activation thresholds.
Interpretation:
Our findings suggest that parathyroid CaSR overactivity can reduce plasma Ca2+ to levels insufficient to activate renal CaSRs, even when an activating mutation is present. These findings identify a conceptually new mechanism of CaSR-dependent Ca2+ balance regulation that aid in explaining the spectrum of hypercalciuria in ADH1 patients.
Funding:
Erasmus+ 2018/E+/4458087, the Canadian Institutes for Health research, the Novo Nordisk Foundation, the Beckett Foundation, the Carlsberg Foundation and Independent Research Fund Denmark.
... 7 The current hypothesized model of transcellular absorption in both humans and rodents consists of apical entry into the enterocyte through the Ca 2þselective channel, transient receptor potential vanilloid 6 (TRPV6), intracellular binding to calbindin-D 9k , and basolateral extrusion via the plasma membrane Ca 2þ -ATPase 1 (PMCA1) or sodium-calcium exchanger. [8][9][10] Interestingly, Trpv6 knockout (KO) or mutant mice (Trpv6 mt ) do not display a severe Ca 2þ wasting phenotype, strongly inferring another apical Ca 2þ entry channel. [10][11][12] Voltage-dependent L-type calcium channel, alpha 1D subunit (Ca v 1.3) has been proposed as a complementary channel to TRPV6. ...
... Murine tissue was taken as previously described, 9 snap frozen in liquid nitrogen, and stored at -80 C until use. At each age, the length of the whole small intestine was measured. ...
Background & aims:
Intestinal Ca2+ absorption early in life is vital to achieving optimal bone mineralization. The molecular details of intestinal Ca2+ absorption have been defined in adults after peak bone mass is obtained, but they are largely unexplored during development. We sought to delineate the molecular details of transcellular Ca2+ absorption during this critical period.
Methods:
Expression of small intestinal and renal calcium transport genes was assessed by using quantitative polymerase chain reaction. Net calcium flux across small intestinal segments was measured in Ussing chambers, including after pharmacologic inhibition or genetic manipulation of TRPV6 or Cav1.3 calcium channels. Femurs were analyzed by using micro-computed tomography and histology.
Results:
Net TRPV6-mediated Ca2+ flux across the duodenum was absent in pre-weaned (P14) mice but present after weaning. In contrast, we found significant transcellular Ca2+ absorption in the jejunum at 2 weeks but not 2 months of age. Net jejunal Ca2+ absorption observed at P14 was not present in either Trpv6 mutant (D541A) mice or Cav1.3 knockout mice. We observed significant nifedipine-sensitive transcellular absorption across the ileum at P14 but not 2 months. Cav1.3 knockout pups exhibited delayed bone mineral accrual, compensatory nifedipine-insensitive Ca2+ absorption in the ileum, and increased expression of renal Ca2+ reabsorption mediators at P14. Moreover, weaning pups at 2 weeks reduced jejunal and ileal Cav1.3 expression.
Conclusions:
We have detailed novel pathways contributing to transcellular Ca2+ transport across the distal small intestine of mice during development, highlighting the complexity of the multiple mechanisms involved in achieving a positive Ca2+ balance early in life.
... 7 The current hypothesized model of transcellular absorption in both humans and rodents consists of apical entry into the enterocyte through the Ca 2þselective channel, transient receptor potential vanilloid 6 (TRPV6), intracellular binding to calbindin-D 9k , and basolateral extrusion via the plasma membrane Ca 2þ -ATPase 1 (PMCA1) or sodium-calcium exchanger. [8][9][10] Interestingly, Trpv6 knockout (KO) or mutant mice (Trpv6 mt ) do not display a severe Ca 2þ wasting phenotype, strongly inferring another apical Ca 2þ entry channel. [10][11][12] Voltage-dependent L-type calcium channel, alpha 1D subunit (Ca v 1.3) has been proposed as a complementary channel to TRPV6. ...
... Murine tissue was taken as previously described, 9 snap frozen in liquid nitrogen, and stored at -80 C until use. At each age, the length of the whole small intestine was measured. ...
Objectives:
Intestinal Ca2+ absorption early in life is vital to achieving optimal bone mineralization. The molecular details of intestinal Ca2+ absorption have been defined in adults, after peak bone mass has been reached, but are largely unexplored during development. We sought to delineate the molecular details of transcellular Ca2+ absorption across the small intestine which facilitate a positive calcium balance during growth.
Methods:
We used wildtype, Cav1.3 knockout and Trpv6 mutant mice. Expression of small intestinal and renal calcium transport genes was assessed using quantitative PCR. Net transcellular 45-calcium flux across intestinal segments was measured in Ussing chambers. Femurs we analyzed using micro-CT and histology.
Results:
Significant TRPV6 mediated Ca2+ flux across the duodenum was absent in pre-weaned (P14) mice but occurred post-weaning. In contrast, we found significant transcellular Ca2+ absorption in the jejunum and ileum at P14 but not 2 months. TRPV6 and Cav1.3 are necessary for this jejunal absorption and Cav1.3 appears to mediate absorption across the ileum although compensation is present in knockout pups. Knockout of Cav1.3 induces a compensatory increase in renal Ca2+ reabsorption in P14 mice although these pups have increased growth plate thickness suggesting delayed bone mineralization.
Conclusions:
This work provides molecular details of how the small intestine facilitates increased demand for Ca2+ early in life to meet the requirements of growth and highlights the complexity of the multiple mechanisms involved in achieving a positive Ca2+ balance.
Funding sources:
This work is funded by grants from the Women and Children's Health Research Institute, supported by the Stollery Children's Hospital Foundation, and the National Sciences and Engineering Research Council to RTA, who is the Canada Research Chair in Renal Epithelial Transport Physiology. MRB is supported by a Vanier Canada Graduate Scholarship, Alberta Innovates Clinician Fellowship and an NSERC Michael Smith Foreign Study Supplement. H. Dimke is funded by the Danish Medical Research Council. Work at UdS was funded by Deutsche Forschungsgemeinschaft (DFG) by IRTG1830 (to JE, VF), Sonderforschungsbereich (SFB) 894 (to JE, PW) and SFB TRR152 (to VF).
... In rabbit and mouse small intestine it has been shown that PMCA1 expression is higher proximally in the duodenum than in the jejunum or ileum, and in the large bowel higher in the cecum and ascending colon than in the descending colon (9, 125). Alexander et al. (9) also found that PMCA1 was the only isoform present in enterocytes throughout human and mouse intestine, whereas PMCA4 was the predominant isoform in smooth muscle layers, increasing in expression distally from small to large bowel. ...
... In contrast to intestinal absorption, recent data suggest that PMCA4 may be the major isoform involved in renal active Ca 2ϩ transport, where it shows strong localization to distal tubular regions in human and mouse kidney (9). In a cultured murine model of the distal convolution, PMCA4, but not PMCA1, was found to be enriched when compared with total cortical expression along with the other components of renal transcellular transport TRPV5, calbindin-D 28k , and NCX1 (381). ...
The Ca²⁺extrusion function of the four mammalian isoforms of the plasma membrane calcium ATPases (PMCAs) is well established. There is also ever-increasing detail known of their roles in global and local Ca²⁺homeostasis and intracellular Ca²⁺signaling in a wide variety of cell types and tissues. It is becoming clear that the spatiotemporal patterns of expression of the PMCAs and the fact that their abundances and relative expression levels vary from cell type to cell type both reflect and impact on their specific functions in these cells. Over recent years it has become increasingly apparent that these genes have potentially significant roles in human health and disease, with PMCAs1-4 being associated with cardiovascular diseases, deafness, autism, ataxia, adenoma, and malarial resistance. This review will bring together evidence of the variety of tissue-specific functions of PMCAs and will highlight the roles these genes play in regulating normal physiological functions and the considerable impact the genes have on human disease.
... Transcellular Ca 2+ transport is also typical of kidney cells. Although, various kidney cells express a diversity of PMCA isoforms [64] it seems that it is the PMCA4 isoform that contributes to renal transepithelial Ca 2+ flux rather than PMCA1 [65]. ...
... Splicing at site A can change distribution of PMCAs in polarized cells (see also Section 3). The x and z splice variants of PMCA 1 and 4 localize mostly to the basolateral membrane compartment of polarized epithelia of lung, intestine and kidney where they can facilitate transcellular Ca 2+ fluxes [65,106]. The w insert at the A splice site is the longest and acts as a strong apical signal. ...
Plasma membrane Ca(2+) ATPases (PMCAs) are intimately involved in the control of intracellular Ca(2+) concentration. They reduce Ca(2+) in the cytosol not only by direct ejection, but also by controlling the formation of inositol-1,4,5-trisphosphate and decreasing Ca(2+) release from the endoplasmic reticulum Ca(2+) pool. In mammals four genes (PMCA1-4) are expressed, and alternative RNA splicing generates more than twenty variants. The variants differ in their regulatory characteristics. They localize into highly specialized membrane compartments and respond to the incoming Ca(2+) with distinct temporal resolution. The expression pattern of variants depends on cell type; a change in this pattern can result in perturbed Ca(2+) homeostasis and thus altered cell function. Indeed, PMCAs undergo remarkable changes in their expression pattern during tumorigenesis that might significantly contribute to the unbalanced Ca(2+) homeostasis of cancer cells. This article is part of a Special Issue entitled: Calcium and Cell Fate edited by Jacques Haiech, Claus Heizmann and Joachim Krebs.
... Exit of calcium across the basolateral membrane occurs via the 3Na + /1Ca ++ exchanger NCX1 and/or the plasma membrane calcium ATPase isoform 4 (PMCA4) [4]. PMCA4 can be detected at the basolateral membrane of DCT/CNT and cortical TAL, macula densa, and early distal tubules. ...
Extracellular fluid calcium concentration must be maintained within a narrow range in order to sustain many biological functions, encompassing muscle contraction, blood coagulation, and bone and tooth mineralization. Blood calcium value is critically dependent on the ability of the renal tubule to reabsorb the adequate amount of filtered calcium. Tubular calcium reabsorption is carried out by various and complex mechanisms in 3 distinct segments: the proximal tubule, the cortical thick ascending limb of the loop of Henle, and the late distal convoluted/connecting tubule. In addition, calcium reabsorption is tightly controlled by many endocrine, paracrine, and autocrine factors, as well as by non-hormonal factors, in order to adapt the tubular handling of calcium to the metabolic requirements. The present review summarizes the current knowledge of the mechanisms and factors involved in calcium handling by the kidney and, ultimately, in extracellular calcium homeostasis. The review also highlights some of our gaps in understanding that need to be addressed in the future.
... In the current transcellular model, calcium enters the epithelial cell via a selective channel, Transient Receptor Potential Vanilloid subfamily member 6 (TRPV6) present in the apical membrane. It is buffered intracellularly by calbindin-D 9k and shuttled to the basolateral membrane, where it is pumped or exchanged out of the cell by the plasma membrane Ca 2+ -ATPase (PMCA1b) or Na + /Ca 2+ exchanger (NCX1) [30][31][32]. The L-type calcium channel, Ca v 1.3 has been postulated to contribute to transcellular intestinal calcium absorption and expression of its gene (Cacna1d) in the colon is equal to or greater than in the small intestine of mice at 10-12 weeks age and human tissue qPCR array [33]. ...
Calcium absorption and secretion can occur along the length of the small and large intestine. To date, the focus of research into intestinal calcium absorption has been the small intestine, the site contributing the majority of intestinal calcium absorption. However, evidence that the colon contributes as much as 10% of enteral calcium transport has been available for decades. Transcellular calcium absorption and bidirectional paracellular calcium flux contributing to either net absorption or secretion have been observed in the colon, depending on the physiological state. Moreover, the calcium transport pathways contributing to colonic absorption or secretion are regulated by a variety of hormones, including calcitriol, plasma calcium and dietary factors, including prebiotics. Herein we review historical and recent research highlighting the role of colonic calcium transport in overall maintenance of calcium balance, and suggest these data are consistent with the colon being a site of significant regulated transepithelial calcium transport.
... A mouse monoclonal antibody, JA9, specifically recognizes the PMCA4 isoform (ab2783, Abcam, Cambridge, UK), 36 which is the main isoform expressed in the DCT/CNT region. 20,21 Polyclonal rabbit anti-β-actin antibody (#Ab8227, Abcam, Cambridge, UK). ...
Background Treatment with the aminoglycoside antibiotic gentamicin can be associated with severe adverse effects, including renal calcium wasting. The underlying mechanism is unknown but it has been proposed to involve activation of the Ca ²⁺ -sensing receptor (CaSR) in the thick ascending limb, which would increase expression of claudin-14 (CLDN14) and limit Ca ²⁺ reabsorption. However, no direct evidence for this hypothesis has been presented.
Methods We studied the effect of gentamicin in vivo using mouse models with impaired Ca ²⁺ reabsorption in the proximal tubule and the thick ascending limb. We used a Cldn14 promoter luciferase-reporter assay to study CaSR activation and investigated the effect of gentamicin on activity of the distal nephron Ca ²⁺ channel transient potential receptor vanilloid 5 (TPRV5), as determined by patch-clamp in HEK293 cells.
Results Gentamicin increased urinary Ca ²⁺ excretion in wild-type mice following acute and chronic administration. This calciuretic effect was unaltered in mice with genetic CaSR overactivation and was present in furosemide-treated animals, whereas the calciuretic effect in Cldn14 -/- mice and mice with impaired proximal tubular Ca ²⁺ reabsorption (claudin-2 [CLDN2]-deficient Cldn2 -/- mice) was equivalent to that of wild-type mice. In vitro , gentamicin failed to activate the CaSR. In contrast, patch-clamp analysis revealed that gentamicin strongly inhibited rabbit and human TRPV5 activity and that chronic gentamicin administration downregulated distal nephron Ca ²⁺ transporters.
Conclusions Gentamicin does not cause hypercalciuria via activation of the CaSR-CLDN14 pathway or by interfering with proximal tubular CLDN2-dependent Ca ²⁺ reabsorption. Instead, gentamicin blocks distal Ca ²⁺ reabsorption by direct inhibition of the Ca ²⁺ channel TRPV5. These findings offer new insights into calcium wasting in patients treated with gentamicin.
... Solute carrier family 4 member 9 membrane ATPase (ATP2B1 also called PMCA1b), along with the Na þ /Ca 2þ exchangers (SLC8A1 or NCX1), performs the final step in transcellular Ca 2 absorption, extruding Ca 2þ from the cell interior to the interstitial space at the basolateral membrane (Stafford et al., 2017). PMCA1 (ATP2B1) is the only isoform present in enterocytes throughout the human or mouse intestine and its expression is higher proximally in the duodenum than in the jejunum or ileum (Alexander et al., 2015); expression levels are positively correlated with both intestinal Ca 2þ absorption and bone mineral density in mice (Replogle et al., 2014). PMCA1b is also the predominant isomer expressed in the chicken intestine (Melancon and DeLuca, 1970;Bar, 2009;Jonchere et al., 2012;Brionne et al., 2014). ...
... The collection of serum, urine, and feces and measurement of electrolytes and calciotropic hormones has been described previously (15). Real-time qPCR, Ca 2+ permeability of intestinal tissue, microcomputed tomography, immunofluorescence staining, and immunoblotting were performed as previously (8,15,18,(34)(35)(36)(37)(38)(39)(40). Refer to SI Appendix, Supplementary Methods for detailed descriptions. ...
Significance
Significant calcium absorption across renal and intestinal epithelia occurs via the paracellular pathway. However, the identity of the paracellular pore involved is unknown. Claudin-2 and claudin-12 contribute paracellular calcium permeability in cell models, but single knockout animals don’t have altered serum calcium or bone mineralization. To investigate this, Cldn2/12 double knockout mice were generated. They display decreased intestinal calcium absorption and renal calcium wasting, resulting in hypocalcemia and markedly reduced bone mineralization. Claudin-2 and claudin-12 don’t physically interact in vitro, and coexpression has an additive effect on calcium permeability. Our work identifies claudin-2 and claudin-12 as important constituents of the paracellular Ca ²⁺ pathway in intestine and kidney enabling calcium transport and highlights their important complementary roles in maintaining calcium homeostasis.
... Solute carrier family 4 member 9 membrane ATPase (ATP2B1 also called PMCA1b), along with the Na þ /Ca 2þ exchangers (SLC8A1 or NCX1), performs the final step in transcellular Ca 2 absorption, extruding Ca 2þ from the cell interior to the interstitial space at the basolateral membrane (Stafford et al., 2017). PMCA1 (ATP2B1) is the only isoform present in enterocytes throughout the human or mouse intestine and its expression is higher proximally in the duodenum than in the jejunum or ileum (Alexander et al., 2015); expression levels are positively correlated with both intestinal Ca 2þ absorption and bone mineral density in mice (Replogle et al., 2014). PMCA1b is also the predominant isomer expressed in the chicken intestine (Melancon and DeLuca, 1970;Bar, 2009;Jonchere et al., 2012;Brionne et al., 2014). ...
The avian egg is a giant reproductive cell protected by an eggshell, which biomineralizes in the uterus, in an acellular fluid that contains ionic and organic precursors. The distinctive features of the eggshell, as compared to bone or teeth, are its mineral composition (CaCO3 in the form of calcite) and the absence of cell-directed assembly during its mineralization on extracellular membranes. In chickens, the hen exports daily 2 g of Ca, which is a challenge for maintenance of calcium homeostasis. Hens develop numerous physiological adaptations for providing shell calcium and carbonate. One feature is the development of labile calcium reserves in medullary bone at sexual maturity. Moreover, the level of intestinal Ca uptake increases sixfold though the action of 1,25(OH)2 D3. The uterine segment of the oviduct acquires the capacity to secrete large amounts of Ca²⁺ and HCO3– ions and to synthesize/secrete eggshell matrix proteins that control the process of shell mineralization and its textural properties. This chapter describes the mechanisms of ion transfer from bone, intestine, and uterus, the process of shell mineralization and their hormonal regulation.
... The calcium-binding protein calbindin-D 28K (Calb28K) is also abundantly expressed in these cells, where it acts as a calcium buffer that can transport calcium from the apical entry site to the basolateral membrane without perturbing cellular calcium signaling (92). The proteins Na 2+ /Ca 2+ exchanger type 1 (NCX1) and plasma membrane Ca 2+ -ATPase 4 (PMCA4) (93,94) are the primary mechanisms mediating calcium extrusion across the basolateral membrane. ...
Nephrolithiasis is a worldwide problem with increasing prevalence, enormous costs, and significant morbidity. Calcium-containing kidney stones are by far the most common kidney stones encountered in clinical practice. Consequently, hypercalciuria is the greatest risk factor for kidney stone formation. Hypercalciuria can result from enhanced intestinal absorption, increased bone resorption, or altered renal tubular transport. Kidney stone formation is complex and driven by high concentrations of calcium-oxalate or calcium-phosphate in the urine. After discussing the mechanism mediating renal calcium salt precipitation, we review recent discoveries in renal tubular calcium transport from the proximal tubule, thick ascending limb, and distal convolution. Furthermore, we address how calcium is absorbed from the intestine and mobilized from bone. The effect of acidosis on bone calcium resorption and urinary calcium excretion is also considered. Although recent discoveries provide insight into these processes, much remains to be understood in order to provide improved therapies for hypercalciuria and prevent kidney stone formation.
Expected final online publication date for the Annual Review of Physiology, Volume 84 is February 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Staining of formalin-fixed paraffin-embedded kidney tissue was done as previously described. 24 Sections were incubated in tissueclear and rehydrated in a series of graded ethanol before heat-induced antigen retrieval using 10 mM Tris and 0.5 mM EGTA buffer (TEG, pH 9.0). Endogenous peroxidase enzymes were blocked using 0.6% H 2 O 2 and free aldehyde groups blocked using 50 mM ). ...
Activation of the basolateral calcium sensing receptor (CaSR) in the renal tubular thick ascending limb (TAL) increases claudin‐14 expression, which reduces paracellular calcium (Ca2+) permeability, thus increasing urinary Ca2+ excretion. However, the upstream signaling pathway contributing to altered CLDN14 gene expression is unknown. To delineate this pathway, we identified and then cloned the CaSR responsive region including the promoter of mouse Cldn14 into a luciferase reporter vector. This 1500 bp sequence upstream of the 5′ UTR of Cldn14 variant 1, conferred increased reporter activity in the presence of high extracellular Ca2+ (5 mM) relative to a lower (0.5 mM) concentration. Assessment of Cldn14 reporter activity in response to increased extracellular Ca2+ in the presence or absence of specific inhibitors confirmed signaling through PLC and p38, but not JNK. Overexpression of SP1 attenuated Cldn14 reporter activity in response to CasR signaling. SP1 is expressed in the TAL and phosphorylation was attenuated by CaSR signaling. Finally, activating mutations in the CaSR increased Cldn14 reporter activity while a dominant negative mutation in the CaSR inhibited it. Together, these studies suggest that basolateral activation of the CASR leads to increased Cldn14 expression via a PLC‐ stimulated p38 pathway that prevents Sp1 mediated repression.
... The collection of serum, urine, and feces and measurement of electrolytes and calciotropic hormones has been described previously (15). Real-time qPCR, Ca 2+ permeability of intestinal tissue, microcomputed tomography, immunofluorescence staining, and immunoblotting were performed as previously (8,15,18,(34)(35)(36)(37)(38)(39)(40). Refer to SI Appendix, Supplementary Methods for detailed descriptions. ...
... In the current transcellular model, calcium enters the epithelial cell via a selective channel, Transient Receptor Potential Vanilloid subfamily member 6 (TRPV6) present in the apical membrane. It is buffered intracellularly by calbindin-D 9k and shuttled to the basolateral membrane, where it is pumped or exchanged out of the cell by the plasma membrane Ca 2+ -ATPase (PMCA1b) or Na + /Ca 2+ exchanger (NCX1) [30][31][32]. The L-type calcium channel, Ca v 1.3 has been postulated to contribute to transcellular intestinal calcium absorption and expression of its gene (Cacna1d) in the colon is equal to or greater than in the small intestine of mice at 10-12 weeks age and human tissue qPCR array [33]. ...
Objectives
Calcium (Ca2+) is a vital micronutrient for many physiological functions with the greatest rate of accumulation occurring during the critical period of infancy. Previous work has demonstrated that molecular mechanisms of intestinal Ca2+ absorption across the small intestine are significantly different in animal models of infants and adults to permit greater absorption early in life. The colon contributes to overall Ca2+ balance in adults via transcellular, TRPV6 mediated and paracellular claudin-2 and -12 mediated pathways. Whether these same colonic pathways contribute to overall Ca2+ absorption in infants is not known. Here we aimed to investigate the molecular details of Ca2+ absorption across the large intestine in murine models of infancy relative to older mice.
Methods
Mice at 14 days (P14) were used as a model of suckling infants and mice at 2 months were employed to represent adult physiology. Wildtype and mutant mice with a non-functioning Trpv6 or deletions of Cldn2 or Cldn12 were used. Net 45Ca2+ flux (JCa) and Ca2+ permeability (PCa) were measured in Ussing chambers. Gene expression was determined by real-time PCR.
Results
JCa indicates net absorption across the colon at both P14 and 2 months. While gene expression of Trpv6 and S100g suggest greater cellular uptake of Ca2+ into colonocytes at P14, net JCa in vitro was not different than at 2 months. In contrast to previous work in mice at 2 months, TRPV6 does not mediate JCa at P14. PCa was 20% greater at P14 than 2 months, suggesting greater capacity for bidirectional diffusion of Ca2+ down an electrochemical gradient in younger mice. In contrast to previous work in mice at 2 months, claudin-2 and claudin-12 do not mediate PCa at P14 and, expression of Cldn2 and Cldn12 were significantly reduced in younger mice.
Conclusions
These results improve our understanding of intestinal Ca2+ handling during a critical age early in life. Future work is required to delineate molecular details under in vivo conditions of colonic Ca2+ transport in infants.
Funding Sources
This work was funded by grants from the Women and Children's Health Research Institute, which is supported by the Stollery Children's Hospital Foundation, and the National Sciences and Engineering Research Council to RTA, who is the Canada Research Chair in Renal Epithelial Transport Physiology.
... The significance of these modulations of renal ATPases cannot be overemphasized, given that Na + ,K + -ATPase is important in controlling the reabsorption of Na + and water in the kidney 109,110 ; while Ca 2+ -ATPase is involved in renal active Ca 2+ transport. 111,112 Consequently, the elevated concentrations of renal calcium and sodium, as well as the lowered chloride, magnesium and potassium, induced by doxorubicin in this study, is reflective of compromised membranes of the renal tissues. However, pretreatment with the extracts prevented the doxorubicin-induced electrolyte imbalance. ...
Background:
The major draw-back of doxorubicin's use in chemotherapy is its toxicity on various organs including the kidneys. This study investigated the potential protective role of aqueous leaf-extracts of Chromolaena odorata and Tridax procumbens against nephrotoxicity induced by doxorubicin.
Methods:
To this end, their impact on plasma biomarkers of kidney function, as well as renal lipid profile, biomarkers of oxidative stress, electrolyte profile and activities of renal ATPases was monitored in doxorubicin treated rats. Metformin (250 mg/kg body weight, orally) and the extracts (50, 75 and 100 mg/kg, orally) were daily administered for 14 days; while nephrotoxicity was induced with doxorubicin (15 mg/kg, intra-peritioneally), once on the 12th day of study.
Results:
The plasma concentrations of creatinine, and urea; as well as the renal malondialdehyde, cholesterol, calcium and sodium concentrations in the Test control, were significantly (P < .05) higher than those of all the other groups. However, the renal concentrations of ascorbic acid, chloride, magnesium and potassium, and the renal activities of catalase, glutathione peroxidase superoxide dismutase, Ca2+-ATPase, Mg2+-ATPase and Na+,K+-ATPase in the Test control were significantly (P < .05) lower than those of all the other groups.
Conclusions:
Pre-treatment with the extracts and metformin boosted endogenous antioxidants, and prevented doxorubicin-induced renal damage, as indicated by the attenuation of doxorubicin-induced renal oxidative stress, as well as the attenuation of doxorubicin-induced adverse alterations in renal cholesterol, ATPases and electrolyte balance, and plasma biomarkers of kidney function, and keeping them at near-normal values.
... Kidneys were snap frozen in liquid nitrogen after isolation and stored at À80 C for future RNA extraction. RNA was extracted from kidneys using TRIzol (Invitrogen, Carlsbad, CA) and processed as previously described (39). Samples were subjected to DNase treatment (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) according to the manufacturer's protocol and reversed transcribed into cDNA (iScript cDNA synthesis kit, Bio-Rad). ...
Variations in the CLDN14 gene have been linked to increased risk of hypercalciuria and kidney stone formation. However, the exact cellular localization of CLDN14 and its regulation remain to be fully delineated. To this end, we generated a novel antibody that allowed the detection of CLDN14 in paraffin-embedded renal sections. This showed CLDN14 to be detectable in the kidney only after induction of hypercalcemia in rodent models. Protein expression in kidney is localized exclusively to the thick ascending limbs (TAL), mainly restricted to the cortical and upper medullary portion of the kidney. However not all cells in the TAL expressed the tight junction protein. In fact, CLDN14 was primarily expressed in cells also expressing CLDN16, but devoid of CLDN10. CLDN14 appeared in very superficial apical cell domains and near cell junctions in a belt-like formation along the apical cell periphery. In transgenic mice, Cldn14 promotor-driven LacZ activity did not show complete colocalization with CLDN14 protein nor was it increased by hypercalcemia, suggesting that LacZ activity cannot be used as a marker for CLDN14 localization and regulation in this model. In conclusion, CLDN14, showed a restricted localization pattern in the apical domain of select cells of the TAL.
... Immunohistochemistry. PBS/heparin-perfused 129S6/SvEvTac mouse kidneys were paraffin-embedded and sectioned every 2 μm prior to formalin-fixation as previously described 48 . Sections were then rehydrated using Tissue-Clear (Tissue-Tek, Dakura sections) and subsequently incubated in graded ethanol solutions into water and next submitted to heat induced antigen retrieval with a TEG solution (10 mM Tris, 0.5 mM EGTA, pH = 9.0). ...
In the renal collecting duct, intercalated cells regulate acid-base balance by effluxing protons through the v-H+-ATPase, and bicarbonate via apical pendrin or the basolateral kidney anion exchanger 1 (kAE1). Additionally, collecting duct cells play an essential role in transepithelial absorption of sodium and chloride. Expression of kAE1 in polarized MDCK I cells was previously shown to decrease trans-epithelial electrical resistance (TEER), suggesting a novel role for kAE1 in paracellular permeability. In our study, we not only confirmed that inducible expression of kAE1 in mIMCD3 cells decreased TEER but we also observed (i) increased epithelial absolute permeability to both sodium and chloride, and (ii) that this effect was dependent on kAE1 activity. Further, kAE1 regulated tight junction properties through the tight junction protein claudin-4, a protein with which it physically interacts and colocalizes. These findings unveil a novel interaction between the junctional protein claudin-4 and the kidney anion exchanger, which may be relevant to ion and/or pH homeostasis.
... For detection, all sections were incubated with EnVision-HRP coupled to goat-anti-rabbit IgG (K4003, Dako) and incubated with AEC + Substrate-Chromogen solution (K3461, Dako). Double immunostaining was performed as previously described68 . After rehydration, sections were boiled in Tris-EGTA (pH 9.0) and incubated in PBS with 0.6% H 2 O 2 /50 mM NH 4 Cl. ...
Non-alcoholic steatohepatitis (NASH) signified by hepatic steatosis, inflammation, hepatocellular injury, and fibrosis is a growing cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma. Hepatic fibrosis resulting from accumulation of extracellular matrix proteins secreted by hepatic myofibroblasts plays an important role in disease progression. Activated hepatic stellate cells (HSCs) have been identified as the primary source of myofibroblasts in animal models of hepatotoxic liver injury; however, so far HSC activation and plasticity have not been thoroughly investigated in the context of NASH-related fibrogenesis. Here we have determined the time-resolved changes in the HSC transcriptome during development of Western diet- and fructose-induced NASH in mice, a NASH model recapitulating human disease. Intriguingly, HSC transcriptional dynamics are highly similar across disease models pointing to HSC activation as a point of convergence in the development of fibrotic liver disease. Bioinformatic interrogation of the promoter sequences of activated genes combined with loss-of-function experiments indicates that the transcriptional regulators ETS1 and RUNX1 act as drivers of NASH-associated HSC plasticity. Taken together, our results implicate HSC activation and transcriptional plasticity as key aspects of NASH pathophysiology.
... Immunohistochemical staining of formalin fixed paraffin embedded tissue was done as described in detail previously [35]. Briefly, sections were rehydrated from xylene through graded ethanol solutions into water and subsequently boiled in TEG buffer (10 mM Tris, 0.5 mM EGTA, pH = 9.0) for heat induced antigen retrieval using. ...
Aldosterone blockade confers substantial cardiovascular and renal protection. The effects of aldosterone on mineralocorticoid receptors (MR) expressed in endothelial cells (EC) within the renal vasculature have not been delineated. We hypothesized that lack of MR in EC may be protective in renal vasculature and examined this by ablating the Nr3c2 gene in endothelial cells (EC-MR) in mice. Blood pressure, heart rate and PAH clearance were measured using indwelling catheters in conscious mice. The role of the MR in EC on contraction and relaxation was investigated in the renal artery and in perfused afferent arterioles. Urinary sodium excretion was determined by use of metabolic cages. EC-MR transgenics had markedly decreased MR expression in isolated aortic endothelial cells as compared to littermates (WT). Blood pressure and effective renal plasma flow at baseline and following AngII infusion was similar between groups. No differences in contraction and relaxation were observed between WT and EC-MR KO in isolated renal arteries during baseline or following 2 or 4 weeks of AngII infusion. The constriction or dilatations of afferent arterioles between genotypes were not different. No changes were found between the groups with respect to urinary excretion of sodium after 4 weeks of AngII infusion, or in urinary albumin excretion and kidney morphology. In conclusion, deletion of the EC-MR does not confer protection towards the development of hypertension, endothelial dysfunction of renal arteries or renal function following prolonged AngII-infusion.
... Staining of PFA-fixed paraffin-embedded tissue was done as previously described with minor modifications ( Alexander et al., 2015;Beggs et al., 2017). Sections were rehydrated in xylene followed by a series of graded ethanol before being subjected to heat-induced antigen retrieval with either 10 mM Tris and 0.5 mM EGTA buffer (pH 9.0). ...
Carbonic anhydrase II (CAII) is expressed along the nephron where it interacts with a number of transport proteins augmenting their activity. Aquaporin-1 (AQP1) interacts with CAII to increase water flux through the water channel. Both CAII and aquaporin-1 are expressed in the thin descending limb (TDL); however, the physiological role of a CAII-AQP1 interaction in this nephron segment is not known. To determine if CAII was required for urinary concentration, we studied water handling in CAII-deficient mice. CAII-deficient mice demonstrate polyuria and polydipsia as well as an alkaline urine and bicarbonaturia, consistent with a type III renal tubular acidosis. Natriuresis and hypercalciuria cause polyuria, however, CAII-deficient mice did not have increased urinary sodium nor calcium excretion. Further examination revealed dilute urine in the CAII-deficient mice. Urinary concentration remained reduced in CAII-deficient mice relative to wild-type animals even after water deprivation. The renal expression and localization by light microscopy of NKCC2 and aquaporin-2 was not altered. However, CAII-deficient mice had increased renal AQP1 expression. CAII associates with and increases water flux through aquaporin-1. Water flux through aquaporin-1 in the TDL of the loop of Henle is essential to the concentration of urine, as this is required to generate a concentrated medullary interstitium. We therefore measured cortical and medullary interstitial concentration in wild-type and CAII-deficient mice. Mice lacking CAII had equivalent cortical interstitial osmolarity to wild-type mice: however, they had reduced medullary interstitial osmolarity. We propose therefore that reduced water flux through aquaporin-1 in the TDL in the absence of CAII prevents the generation of a maximally concentrated medullary interstitium. This, in turn, limits urinary concentration in CAII deficient mice.
... Polymerase Chain Reaction (PCR) -All PCR primers are described in either Tabel-1. The mouse primers for PMCA 1 through 4 (26,27) and PMCA-4a and PMCA-4a/b (28) have been described. The human PMCA primers were designed using Primer Blast from NIH. Human cDNA was the kind gift of Guy Groblewski of the University of Wisconsin. ...
Acute pancreatitis is a disease associated with inflammation and tissue damage. One protein that protects against acute injury, including ischemic injury to both the kidney and heart, renalase, which is secreted into the blood by the kidney and other tissues. However, whether renalase reduces acute injury associated with pancreatitis is unknown. Here, we used both in vitro and in vivo murine models of acute pancreatitis to study renalase effects on this condition. In isolated pancreatic lobules, pretreatment with recombinant human renalase (rRNLS) blocked zymogen activation caused by cerulein, carbachol, and a bile acid. Renalase also blocked cerulein-induced cell injury and histological changes. In the in vivo cerulein model of pancreatitis, genetic deletion of renalase resulted in more severe disease, and administering rRNLS to cerulein-exposed WT mice after pancreatitis onset was protective. Because pathological increases in acinar cell cytosolic calcium levels are central to the initiation of acute pancreatitis, we also investigated whether rRNLS could function through its binding protein, calcium ATPase 4b (PMCA4b), which excretes calcium from cells. We found that PMCA4b is expressed in both murine and human acinar cells and that a PMCA4b selective inhibitor worsens pancreatitis-induced injury and blocked the protective effects of rRNLS. These findings suggest that renalase is a protective plasma protein that reduces acinar cell injury through a plasma membrane calcium ATPase. Since exogenous rRNLS reduces the severity of acute pancreatitis, it has the potential as a therapeutic agent.
... PMCAs belongs to the p-type ATPase family and can form a high-energy phosphorylated intermediate in the reaction cycle. Phosphorylated PMCAs exist in two conformational states, E1 and E2, each being alternative for the other (16). PMCAs in mammals are encoded by four genes that encode PMCA1-4, and are located on human chromosomes 12, 3, 1 and X. ...
The aim of the present study was to investigate the possible mechanism of Janus kinase (JAK)-signal transduction and activator of transcription (STAT)/PSMβ3 signaling in the occurrence of asthenozoospermia. We examined seminal fluid samples from 30 cases of asthenozoospermia and 30 healthy controls. Sperm was collected using the Percoll density gradient centrifugation method. The expression of JAK, STAT and PSMβ3 mRNA was assessed by reverse-transcription quantitative PCR and the protein levels of p-JAK, p-STAT and PSMβ3 were measured by western blot analysis. The PSMβ3 mRNA and protein expression levels were also measured after application of a JAK inhibitor, AG-490, to the control group, with a FITC-labeled monoclonal rabbit anti-human PSMβ3 primary antibody. The cells were observed under a laser confocal microscope. The mRNA levels of JAK, STAT and PSMβ3 in asthenozoospermia were decreased significantly (P<0.05). The protein levels of p-JAK, p-STAT and PSMβ3 in asthenozoospermia were also reduced and the differences were statistically significant (P<0.05). The PSMβ3 mRNA and protein expression levels were decreased in the control group after treatment with the JAK inhibitor, and levels were approximately equal to those of the asthenozoospermia group. PSMβ3 was mainly expressed in round-headed sperm, and less in asthenozoospermia. In conclusion, the JAK-STAT/PSMβ3 signaling transduction pathway may be involved in the pathogenic mechanism of asthenozoospermia.
... 44 The calcium binding protein calbindin-D 28K buffers ionized calcium, which is extruded across the basolateral membrane via calcium dependent ATPases (plasma membrane Ca 2+ ATPase) or a sodium calcium exchanger. 45,46 Micropuncture studies demonstrate that calcium reabsorption from the distal nephron is inhibited by metabolic acidosis, independent of changes in parathyroid hormone (PTH), inferring a direct effect on renal transport processes. 41,47,48 Consistent with this, renal Trpv5 expression is decreased by systemic acid loads and metabolic alkalosis increases expression. ...
Metabolic acidosis is associated with increased urinary calcium excretion and related sequelae, including nephrocalcinosis and nephrolithiasis. The increased urinary calcium excretion induced by metabolic acidosis predominantly results from increased mobilization of calcium out of bone and inhibition of calcium transport processes within the renal tubule. The mechanisms whereby acid alters the integrity and stability of bone have been examined extensively in the published literature. Here, after briefly reviewing this literature, we consider the effects of acid on calcium transport in the renal tubule and then discuss why not all gene defects that cause renal tubular acidosis are associated with hypercalciuria and nephrocalcinosis.
A higher concentration of calcium in breast milk than blood favors paracellular calcium absorption enabling growth during postnatal development. We aimed to determine whether suckling animals have greater intestinal calcium permeability to maximize absorption and to identify the underlying molecular mechanism. We examined intestinal claudin expression at different ages in mice and in human intestinal epithelial (Caco-2) cells in response to hormones or human milk. We also measured intestinal calcium permeability in wildtype, Cldn2 and Cldn12 KO mice and Caco-2 cells in response to hormones or human milk. Bone mineralization in mice was assessed by μCT. Calcium permeability across the jejunum and ileum of mice were 2-fold greater at 2 wk than 2 mo postnatal age. At 2 wk, Cldn2 and Cldn12 expression were greater, but only Cldn2 KO mice had decreased calcium permeability compared to wildtype. This translated to decreased bone volume, cross-sectional thickness, and tissue mineral density of femurs. Weaning from breast milk led to a 50% decrease in Cldn2 expression in the jejunum and ileum. Epidermal growth factor (EGF) in breast milk specifically increased only CLDN2 expression and calcium permeability in Caco-2 cells. These data support intestinal permeability to calcium, conferred by claudin-2, being greater in suckling mice and being driven by EGF in breast milk. Loss of the CLDN2 pathway leads to suboptimal bone mineralization at 2 wk of life. Overall, EGF-mediated control of intestinal claudin-2 expression contributes to maximal intestinal calcium absorption in suckling animals.
Central to the maintenance of calcium homeostasis is the regulated reabsorption of calcium along the nephron. To this end, parathyroid hormone (PTH) is released from the parathyroid gland in response to lowered plasma calcium levels. This hormone acts through the PTH 1 receptor along the nephron to increase urinary phosphate excretion and decrease urinary calcium excretion. In the proximal tubule, PTH inhibits phosphate reabsorption by reducing the abundance of sodium phosphate cotransporters in the apical membrane. PTH likely decreases calcium reabsorption from the proximal tubule, by reducing the reabsorption of sodium, an event necessary for the paracellular movement of calcium across this segment. In the thick ascending limb (TAL), PTH increases calcium permeability and may increase the electrical driving force thereby increasing calcium reabsorption in the TAL. Finally, in the distal convolution, PTH acts to increase transcellular calcium reabsorption by increasing the activity and abundance of the apically expressed calcium channel TRPV5.
Gitelman syndrome (GS) is an autosomal recessive salt-losing tubulopathy caused by biallelic inactivating mutations in the SLC12A3 gene. This gene encodes the thiazide-sensitive sodium-chloride cotransporter (NCC) which is exclusively expressed in the distal convoluted tubules (DCT). GS patients classically present with hypokalemic metabolic alkalosis with hypocalciuria and hypomagnesemia. While hypokalemia and metabolic alkalosis are easily explained by effects of the genotypic defect in GS, the mechanisms by which hypomagnesemia and hypocalciuria develop in GS are poorly understood. In this review, we aim to achieve three major objectives. First, present a concise discussion about current understanding on physiologic calcium and magnesium handling in the DCT. Second, integrate expression data from studies on calciotropic and magnesiotropic proteins relevant to the GS disease state. Lastly, provide insights into the possible mechanisms of calcium-magnesium crosstalk relating to the co-occurrence of hypocalciuria and hypomagnesemia in GS models. Our analyses highlight specific areas of study that are valuable in elucidating possible molecular pathways of hypocalciuria and hypomagnesemia in GS.
The loop of Henle plays a variety of important physiological roles through the concerted actions of ion transport systems in both its apical and basolateral membranes. It is involved most notably in extracellular fluid volume and blood pressure regulation as well as Ca²⁺, Mg²⁺, and acid-base homeostasis because of its ability to reclaim a large fraction of the ultrafiltered solute load. This nephron segment is also involved in urinary concentration by energizing several of the steps that are required to generate a gradient of increasing osmolality from cortex to medulla. Another important role of the loop of Henle is to sustain a process known as tubuloglomerular feedback through the presence of specialized renal tubular cells that lie next to the juxtaglomerular arterioles. This article aims at describing these physiological roles and at discussing a number of the molecular mechanisms involved. It will also report on novel findings and uncertainties regarding the realization of certain processes and on the pathophysiological consequences of perturbed salt handling by the thick ascending limb of the loop of Henle. Since its discovery 150 years ago, the loop of Henle has remained in the spotlight and is now generating further interest because of its role in the renal-sparing effect of SGLT2 inhibitors. © 2022 American Physiological Society. Compr Physiol 12:1-21, 2022.
The transient receptor potential vanilloid 6 (TRPV6) channel is highly Ca²⁺-selective and has been implicated in mediating transcellular Ca²⁺ transport and thus maintaining the Ca²⁺ balance in the body. To characterize its physiological function(s), a detailed expression profile of the TRPV6 channel throughout the body is essential. Capitalizing on a recently established murine Trpv6-reporter strain, we identified primary TRPV6 channel-expressing cells in an organism-wide manner. In a complementary experimental approach, we characterized TRPV6 expression in different tissues of wild-type mice by TRPV6 immunoprecipitation (IP) followed by mass spectrometry analysis and correlated these data with the reporter gene expression. Taken together, we present a TRPV6 expression atlas throughout the entire body of juvenile and adult mice, providing a novel resource to investigate the role of TRPV6 channels in vivo.
Functional properties of the paracellular pathway depend critically on the set of claudins expressed at the tight junction. Two syndromes are causally linked to loss-of-function mutations of claudins: HELIX syndrome caused by genetic variations in the CLDN10 gene, and Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis caused by genetic variations in the CLDN16 or the CLDN19 gene. All three genes are expressed in the kidney, particularly in the thick ascending limb (TAL). However, localization of these claudins in humans and rodents remains to be delineated in detail. We studied the segmental and subcellular expression of CLDN10, 16 and 19 in both paraffin-embedded and frozen kidney sections from adult human, mouse and rat, using immunohistochemistry and immunofluorescence, respectively. Here CLDN10 was present in a subset of medullary and cortical TAL cells, localizing to basolateral domains and tight junction in human and rodent kidney. A weak expression was detected at the tight junction of proximal tubular cells. CLDN16 was primarily expressed in a subset of TAL cells in cortex and outer stripe of outer medulla, restricted to basolateral domains and tight junctional structures in both human and rodent kidney. CLDN19 predominantly colocalized with CLDN16 in tight junctions and basolateral domains of TAL but was also found in basolateral and junctional domains in more distal sites. CLDN10 expression at tight junction almost never overlapped with that of CLND16 and CLDN19, consistent with distinct junctional pathways with different permeation profiles in both human and rodent kidney.
The vacuolar-type H+-ATPase B1 subunit is heavily expressed in the intercalated cells of the collecting system, where it contributes to H+ transport, but has also been described in other segments of the renal tubule. This study aims to determine the localization of the B1 subunit of the vacuolar-type H+-ATPase in the early distal nephron, encompassing thick ascending limbs (TAL) and distal convoluted tubules (DCT) in human kidney and determine if the localization differs between rodents and humans. Antibodies directed against the H+-ATPase B1 subunit were used to determine its localization in paraffin embedded formalin fixed mouse, rat, and human kidneys by light microscopy and in sections of lowicryl embedded rat kidneys by electron microscopy. Abundant H+-ATPase B1 subunit immunoreactivity was observed in the human kidney. As expected, intercalated cells showed the strongest signal, but significant signal was also observed in apical membrane domains of the distal nephron, including TAL, macula densa and DCT. In mouse and rat, H+-ATPase B1 subunit expression could also be detected in apical membrane domains of these segments. In rat, electron microscopy revealed that the H+-ATPase B1 subunit was located in the apical membrane. Furthermore, the H+-ATPase B1 subunit colocalized with other H+-ATPase subunits in the TAL and DCT. In conclusion, the B1 subunit is expressed in the early distal nephron. The physiological importance of H+-ATPase expression in these segments remains to be delineated in detail. The phenotype of disease causing mutations in the B1-subunit may also relate to its presence in the TAL and DCT.
The expression of the CII splice variant of the plasma membrane Ca2+ ATPase 4 (PMCA4) was down-regulated in granule neurons when they were cultured under conditions of partial membrane depolarization
(25 mm KCl), which are required for long term in vitro survival of the neurons. These conditions, which cause a chronic increase of the resting free Ca2+ concentration in the neurons, have recently been shown to promote up-regulation of the PMCA2, 3, and 1CII isoforms. Whereas
the chronic, i.e. >3 days, Ca2+ increase was necessary for the up-regulation of the PMCA1CII, 2, and 3, the down-regulation of the PMCA4CII mRNA was already
evident 1–2 h after the start of culturing in 25 mm KCl. The immunosuppressant calcineurin inhibitor FK506 inhibited the down-regulation of the PMCA4CII at both the protein
and the mRNA level but did not affect the changes of the other PMCA pumps. Direct evidence for the involvement of calcineurin
in the down-regulation of the PMCA4CII was obtained by overexpressing a truncated, constitutively active, and Ca2+-independent form of calcineurin; under these conditions, depolarization was not required for the down-regulation of the PMCA4CII
pump. De novosynthesis of (transcription) factors was required for the down-regulation of the PMCA4CII mRNA. Calcineurin, therefore, controls
the neuronal transcription of PMCA4CII, a splice variant of the pump isoforms that is found almost exclusively in brain.
Intestinal calcium (Ca(2+)) absorption occurs via paracellular and transcellular pathways. While the transcellular route has been extensively studied, mechanisms mediating paracellular absorption are largely unexplored. Unlike passive diffusion, secondarily active paracellular Ca(2+) uptake occurs against an electrochemical gradient with water flux providing the driving force. Water movement is dictated by concentration differences that are largely determined by Na(+) fluxes. Consequently, we hypothesized that Na(+) absorption mediates Ca(2+) flux. NHE3 is central to intestinal Na(+) absorption. NHE3 knockout mice (NHE3(-/-)) display impaired intestinal Na+, water and Ca(2+) absorption. However, the mechanism mediating this latter abnormality is not clear. To investigate this, we used Ussing chambers to measure net Ca(2+) absorption across different segments of wild-type mouse intestine. The caecum was the only segment with net Ca(2+) absorption. Quantitative RT-PCR measurements revealed caecal expression of all genes implicated in intestinal Ca(2+) absorption, including NHE3. We therefore employed this segment for further studies. Inhibition of NHE3 with 100 µM 5-(N-Ethyl-N-isopropyl) amiloride decreased luminal to serosal and increased serosal to luminal Ca(2+) flux. NHE3(-/-) mice had a >60% decrease in luminal to serosal Ca(2+) flux. Ussing chambers experiments under altered voltage clamps (-25, 0, +25 mV) showed decreased transcellular and secondarily active paracellular Ca(2+) absorption in NHE3(-/-) mice relative to wild-type animals. Consistent with this, caecal Trpv6 expression was diminished in NHE3(-/-) mice. Together these results implicate NHE3 in intestinal Ca(2+) absorption and support the theory that this is, at least partially, due to the role of NHE3 in Na(+) and water absorption.
The precise regulation of Ca(2+) dynamics is crucial for proper differentiation and function of osteoclasts. Here we show the involvement of plasma membrane Ca(2+) ATPase (PMCA) isoforms 1 and 4 in osteoclastogenesis. In immature/undifferentiated cells, PMCAs inhibited receptor activator of NF-κB ligand-induced Ca(2+) oscillations and osteoclast differentiation in vitro. Interestingly, nuclear factor of activated T cell c1 (NFATc1) directly stimulated PMCA transcription, whereas the PMCA-mediated Ca(2+) efflux prevented NFATc1 activation, forming a negative regulatory loop. PMCA4 also had an anti-osteoclastogenic effect by reducing NO, which facilitates preosteoclast fusion. In addition to their role in immature cells, increased expression of PMCAs in mature osteoclasts prevented osteoclast apoptosis both in vitro and in vivo. Mice heterozygous for PMCA1 or null for PMCA4 showed an osteopenic phenotype with more osteoclasts on bone surface. Furthermore, PMCA4 expression levels correlated with peak bone mass in premenopausal women. Thus, our results suggest that PMCAs play important roles for the regulation of bone homeostasis in both mice and humans by modulating Ca(2+) signaling in osteoclasts.
Tight regulation of calcium levels is required for many critical biological functions. The Ca2+-sensing receptor (CaSR) expressed by parathyroid cells controls blood calcium concentration by regulating parathyroid hormone (PTH) secretion. However, CaSR is also expressed in other organs, such as the kidney, but the importance of extraparathyroid CaSR in calcium metabolism remains unknown. Here, we investigated the role of extraparathyroid CaSR using thyroparathyroidectomized, PTH-supplemented rats. Chronic inhibition of CaSR selectively increased renal tubular calcium absorption and blood calcium concentration independent of PTH secretion change and without altering intestinal calcium absorption. CaSR inhibition increased blood calcium concentration in animals pretreated with a bisphosphonate, indicating that the increase did not result from release of bone calcium. Kidney CaSR was expressed primarily in the thick ascending limb of the loop of Henle (TAL). As measured by in vitro microperfusion of cortical TAL, CaSR inhibitors increased calcium reabsorption and paracellular pathway permeability but did not change NaCl reabsorption. We conclude that CaSR is a direct determinant of blood calcium concentration, independent of PTH, and modulates renal tubular calcium transport in the TAL via the permeability of the paracellular pathway. These findings suggest that CaSR inhibitors may provide a new specific treatment for disorders related to impaired PTH secretion, such as primary hypoparathyroidism.
Passive paracellular proximal tubular (PT) and intestinal calcium (Ca(2+)) fluxes have been linked to active sodium (re)absorption. Although the epithelial sodium/proton exchanger, NHE3, mediates apical sodium entry at both these sites, its role in Ca(2+) homeostasis remains unclear. We, therefore, set out to determine whether NHE3 is necessary for Ca(2+) (re)absorption from these epithelia by comparing Ca(2+) handling between wild-type and NHE3(-/-) mice. Serum Ca(2+) and plasma parathyroid hormone levels were not different between groups. However, NHE3(-/-) mice had increased serum 1,25-dihydroxyvitamin D(3). The fractional excretion of Ca(2+) was also elevated in NHE3(-/-) mice. Paracellular Ca(2+) flux across confluent monolayers of a PT cell culture model was increased by an osmotic gradient equivalent to that generated by NHE3 across the PT in vivo and by overexpression of NHE3.( 45)Ca(2+) uptake after oral gavage and flux studies in Ussing chambers across duodenum of wild-type and NHE3(-/-) mice confirmed decreased Ca(2+) absorption in NHE3(-/-) mice compared with wild-type mice. Consistent with this, intestinal calbindin-D(9K), claudin-2, and claudin-15 mRNA expression was decreased. Microcomputed tomography analysis revealed a perturbation in bone mineralization. NHE3(-/-) mice had both decreased cortical bone mineral density and trabecular bone mass. Our results demonstrate significant alterations of Ca(2+) homeostasis in NHE3(-/-) mice and provide a molecular link between Na(+) and Ca(2+) (re)absorption.
Calcitonin (CT) is known to affect renal Ca(2+) handling. However, it remains unclear how CT affects Ca(2+) transport in the distal convolutions. The aim of this study was to investigate the contribution of the renal epithelial Ca(2+) channel, transient receptor potential vanilloid 5 (TRPV5), to renal Ca(2+) handling in response to CT.
C57BL/6 mice received a single overnight (16 hr) injection of CT. In addition, TRPV5 knockout (TRPV5(-/-)) mice and their wild type (TRPV5(+/+)) controls, received three bolus injections of CT over a 40 hr study period. All experimental groups were placed in metabolic cages.
C57BL/6 mice received a single bolus injection of CT, which significantly reduced the urinary Ca(2+) excretion. In addition, urinary Na(+) and K(+) excretion also decreased after CT administration. No apparent changes in renal expression of TRPV5, calbindin-D(28K) (CaBP28K) or TRPV6 could be detected between CT- and vehicle-treated mice. To evaluate whether TRPV5 activity is needed for the CT-induced increase in Ca(2+) reabsorption, mice with genetic ablation of TRPV5 (TRPV5(-/-)) were employed. TRPV5(-/-) mice as well as their wild-type (TRPV5(+/+)) controls received three bolus injections of CT over a 40-hr study period. Overnight (16 hrs) as well as the subsequent 24-hr urine was collected. Overnight urinary Ca(2+) excretion was reduced in both TRPV5(-/-) and TRPV5(+/+) mice after a bolus injection of CT. The subsequent 24-hr urinary excretion of Ca(2+) which was collected after the third bolus injection showed no effect of CT on renal Ca(2+) handling in either mice group. Accordingly, CT did not alter the intrarenal protein abundance of TRPV5 and CaBP28K after three bolus injections of CT.
CT augments the renal reabsorptive capacity for Ca(2+). This increase is likely to occur independently of TRPV5.
The kidney plays an important role in maintaining the systemic Ca2+ and Mg2+ balance. Thus the renal reabsorptive capacity of these cations can be amended to adapt to disturbances in plasma Ca2+ and Mg2+ concentrations. The reabsorption of Ca2+ and Mg2+ is driven by transport of other electrolytes, sometimes through selective channels and often supported by hormonal stimuli. It is, therefore, not surprising that monogenic disorders affecting such renal processes may impose a shift in, or even completely blunt, the reabsorptive capacity of these divalent cations within the kidney. Accordingly, in Dent's disease, a disorder with defective proximal tubular transport, hypercalciuria is frequently observed. Dysfunctional thick ascending limb transport in Bartter's syndrome, familial hypomagnesaemia with hypercalciuria and nephrocalcinosis, and diseases associated with Ca2+-sensing receptor defects, markedly change tubular transport of Ca2+ and Mg2+. In the distal convolutions, several proteins involved in Mg2+ transport have been identified [TRPM6 (transient receptor potential melastatin 6), proEGF (pro-epidermal growth factor) and FXYD2 (Na+/K+-ATPase gamma-subunit)]. In addition, conditions such as Gitelman's syndrome, distal renal tubular acidosis and pseudohypoaldosteronism type II, as well as a mitochondrial defect associated with hypomagnesaemia, all change the renal handling of divalent cations. These hereditary disorders have, in many cases, substantially increased our understanding of the complex transport processes in the kidney and their contribution to the regulation of overall Ca2+ and Mg2+ balance.
Regulation of the plasma membrane Ca2+ pump in the cell is of critical importance in maintaining calcium homeostasis. Since protein kinase C is known to regulate functions of cellular proteins by direct phosphorylation or by inducing their gene expression, we investigated the possible involvement of protein kinase C in the regulation of the plasma membrane Ca2+ pump. The Ca2+ pump was isolated by immunoprecipitation from [32P]orthophosphate-labeled cultured rat aortic endothelial cells grown in the absence or presence of phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. PMA treatment of cells led to a rapid increase in the phosphorylation level (1.3-fold) within 5 min and a further increase to 2.9-fold after 3 h. Prolonged PMA treatment also induced the accumulation of the Ca2+ pump mRNA, followed by increased levels of the pump protein. The peak level of the pump mRNA induction occurred at 4 h and was 8-20-fold higher than the control culture without PMA. The rate of the Ca2+ pump protein accumulation was slower, reaching a maximum of 3.5-fold after 6 h. Induction of the pump mRNA was suppressed by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and by down-regulation of protein kinase C. Inactive phorbol ester 4 alpha-phorbol didecanoate also failed to mimic the PMA effect. These results suggest that the induction of Ca2+ pump expression is mediated by a protein kinase C-dependent mechanism. Furthermore, since the induction of the Ca2+ pump mRNA was blocked when cycloheximide and PMA were added together, this suggests that newly synthesized protein factor is needed to produce the mRNA induction. Our results suggest that protein kinase C is involved in the regulation of the Ca2+ pump in endothelial cells. At the protein level, it modifies the Ca2+ pump by phosphorylation, and at the gene level, it stimulates the expression of its mRNA and thereby increases the amount of the pump protein.
Human calcium transporting tissues were examined to determine whether they contained a protein similar to the Ca++-Mg++ adenosine triphosphatase (Ca++-Mg++ATPase) pump of the human erythrocyte membrane. Tissues were processed for immunoperoxidase staining using monoclonal antibodies against purified Ca++-Mg++ATPase. In human kidneys, specific staining was found only along the basolateral membrane of the distal convoluted tubules. Glomeruli and other segments of the nephron did not stain. Staining of erythrocytes in human spleen was readily observed. Human small intestine, human parathyroid, and human liver showed no antigens that crossreacted with the antibodies to Ca++-Mg++ATPase. Specific staining of distal tubule basolateral membranes from the kidney of a chimpanzee was also noted. Our experiments show, for the first time, that basolateral membranes of the human distal convoluted tubule contain a protein that is immunologically similar to the human erythrocyte Ca++-Mg++ATPase. These observations suggest that the cells of the distal convoluted tubules of human kidney may have a calcium pump similar to that of human erythrocyte membranes.
A complementary DNA for a third isoform of the calmodulin-sensitive plasma membrane Ca-ATPase has been isolated from a rat brain cDNA library. The nucleotide sequence of the 5.1-kilobase pair cDNA has been determined, and the amino acid sequence of the protein, designated PMCA3, has been deduced. PMCA3 is 1159 amino acids in length and has an Mr of 127,300. It exhibits 81% and 85% amino acid identity, respectively, to isoforms 1 and 2 (PMCA1 and PMCA2) of the plasma membrane Ca-ATPase. The transcript encoding PMCA3 is similar to that of PMCA1 in that it contains a sequence in the 3'-untranslated region that has the potential to encode an alternative calmodulin binding domain and carboxyl terminus. The tissue distribution of mRNAs encoding isoforms 1, 2, and 3 has been determined by Northern blot hybridization analyses. PMCA1 mRNAs are expressed in all tissues examined, suggesting that this protein may serve as a housekeeping form of the enzyme. However, PMCA2 and PMCA3 mRNAs exhibit a high degree of tissue specificity. PMCA2 mRNAs are expressed predominantly in brain and heart, whereas PMCA3 mRNAs are expressed predominantly in brain and skeletal muscle.
Antibodies against the four isoforms of the human plasma membrane Ca²⁺-ATPase (PMCA) were raised using an N-terminal sequence of the pump as epitope. The antibodies against PMCA isoforms 1, 2, and 3 were not species-specific, e.g. they also recognized the corresponding proteins in rat, whereas that against the human PMCA isoform 4 failed to do so.
The tissue distribution of the four isoforms was estimated by Western blot analysis. Two, PMCA1 and PMCA4, were expressed in all tissues tested (with the exception of the choroid plexus, where the former was not detected). In most tissues the signal from the PMCA1 protein exceeded that of PMCA4, the exception being the erythrocyte. The PMCA2 and PMCA3 proteins were only found in neuronal tissues; the PMCA2 protein was present in high concentrations in the cerebellum and in the cerebral cortex. At variance with previous results on mRNA (e.g. the kidney) no other tissues contained the PMCA2 protein. PMCA3 was the other tissue-specific isoform; in agreement with results in the rat, the protein was found in human neuronal tissues, particularly in the choroid plexus, but was practically absent in all other tissues tested.
The epitope location and specificity of monoclonal antibodies JA9, 5F10 and JA3, raised against the human plasma membrane Ca2+ pump (hPMCA), were analysed by using synthetic peptides of the corresponding epitopes as well as the complete isoforms, hPMCA4b, hPMCA4a and hPMCA1b, expressed in COS-1 cells. The experiments with the peptides showed that JA9 reacted specifically with a region containing residues 51-75 of hPMCA4 (a or b), but not with the same region of isoforms 1, 2 or 3. JA3 reacted with residues 1156-1180, a region unique to hPMCA4b. 5F10 reacted in the region of residues 719-738, which is highly conserved in all PMCA isoforms. Indeed, 5F10 recognized all three isoforms expressed in COS-1 cells. JA9, in contrast, reacted with both variants a and b of hPMCA4 but not with hPMCA1, and JA3 recognized exclusively hPMCA4b. We used these antibodies to discern the distribution of hPMCA4a and hPMCA4b in human brain, heart, kidney and lung. In Western blots of human brain samples, we could identify both hPMCA4a and hPMCA4b. Heart tissue also showed isoform 4b, and probably 4a. In contrast, kidney and lung showed primarily hPMCA4b. In brain, overlapping bands that did not correspond to either variant of hPMCA4 were detected, and in kidney a band migrating in the same position as hPMCA1b was observed. The distribution of the a and b forms of hPMCA4 at the protein level, as analysed by these antibodies, is consistent with the available data about the abundance of mRNAs for the hPMCA isoforms. The presence of hPMCA4b in all the samples supports the proposed role of this isoenzyme as a constitutive form of the pump.
Plasma membrane Ca2+ ATPase (PMCA) pump isoforms 2, 3, and 1CII are expressed in large amounts in the cerebellum of adult rats but only minimally in neonatal cerebellum. These isoforms were almost undetectable in rat neonatal cerebellar granule cells 1-3 days after plating, but they became highly expressed after 7-9 days of culturing under membrane depolarizing conditions (25 mM KCl). The behavior of isoform 4 was different: it was clearly detectable in adult cerebellum but was down-regulated by the depolarizing conditions in cultured cells. 25 mM KCl-activated L-type Ca2+ channels, significantly increasing cytosolic Ca2+. Changes in the concentration of Ca2+ in the culturing medium affected the expression of the pumps. L-type Ca2+ channel blockers abolished both the up-regulation of the PMCA1CII, 2, and 3 isoforms and the down-regulation of PMCA4 isoform. When granule cells were cultured in high concentrations of N-methyl-D-aspartic acid, a condition that increased cytosolic Ca2+ through the activation of glutamate-operated Ca2+ channels, up-regulation of PMCA1CII, 2, and 3 and down-regulation of PMCA4 was also observed. The activity of the isoforms was estimated by measuring the phosphoenzyme intermediate of their reaction cycle: the up-regulated isoforms, the activity of which was barely detectable at plating time, accounted for a large portion of the total PMCA activity of the cells. No up-regulation of the sarcoplasmic/endoplasmic reticulum calcium pump was induced by the depolarizing conditions.
Calcium pumps of the plasma membrane (also known as plasma membrane Ca(2+)-ATPases or PMCAs) are responsible for the expulsion of Ca(2+) from the cytosol of all eukaryotic cells. Together with Na(+)/Ca(2+) exchangers, they are the major plasma membrane transport system responsible for the long-term regulation of the resting intracellular Ca(2+) concentration. Like the Ca(2+) pumps of the sarco/endoplasmic reticulum (SERCAs), which pump Ca(2+) from the cytosol into the endoplasmic reticulum, the PMCAs belong to the family of P-type primary ion transport ATPases characterized by the formation of an aspartyl phosphate intermediate during the reaction cycle. Mammalian PMCAs are encoded by four separate genes, and additional isoform variants are generated via alternative RNA splicing of the primary gene transcripts. The expression of different PMCA isoforms and splice variants is regulated in a developmental, tissue- and cell type-specific manner, suggesting that these pumps are functionally adapted to the physiological needs of particular cells and tissues. PMCAs 1 and 4 are found in virtually all tissues in the adult, whereas PMCAs 2 and 3 are primarily expressed in excitable cells of the nervous system and muscles. During mouse embryonic development, PMCA1 is ubiquitously detected from the earliest time points, and all isoforms show spatially overlapping but distinct expression patterns with dynamic temporal changes occurring during late fetal development. Alternative splicing affects two major locations in the plasma membrane Ca(2+) pump protein: the first intracellular loop and the COOH-terminal tail. These two regions correspond to major regulatory domains of the pumps. In the first cytosolic loop, the affected region is embedded between a putative G protein binding sequence and the site of phospholipid sensitivity, and in the COOH-terminal tail, splicing affects pump regulation by calmodulin, phosphorylation, and differential interaction with PDZ domain-containing anchoring and signaling proteins. Recent evidence demonstrating differential distribution, dynamic regulation of expression, and major functional differences between alternative splice variants suggests that these transporters play a more dynamic role than hitherto assumed in the spatial and temporal control of Ca(2+) signaling. The identification of mice carrying PMCA mutations that lead to diseases such as hearing loss and ataxia, as well as the corresponding phenotypes of genetically engineered PMCA "knockout" mice further support the concept of specific, nonredundant roles for each Ca(2+) pump isoform in cellular Ca(2+) regulation.
Calcium transport across a monolayer of Madin-Darby canine kidney (MDCK) cells was measured in response to stimulation of the basal surface with calcium-sensing receptor (CaR) agonists. Stimulation of the CaR resulted in a time- and concentration-dependent inhibition of calcium transport but did not change transepithelial voltage or resistance. Inhibition of transport was not altered by pretreatment of cells with pertussis toxin but was blocked by the phospholipase C (PLC) inhibitor U-73122. To determine a potential mechanism by which the CaR could inhibit calcium transport, we measured activity of the plasma membrane calcium ATPase (PMCA). Stimulation of the CaR on the basal surface resulted in an inhibition of the PMCA in a concentration- and PLC-dependent manner. Thus stimulation of the CaR inhibits both calcium transport and PMCA activity through a PLC-dependent pathway. These studies provide the first direct evidence that calcium can inhibit its own transcellular absorption in a model of the distal tubule. In addition, they provide a potential mechanism for the CaR to inhibit calcium transport, inhibition of PMCA.
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.
Extracellular nucleotides are important regulators of epithelial ion transport. Here we investigated nucleotide-mediated effects on colonic NaCl secretion and the signal transduction mechanisms involved. Basolateral UDP induced a sustained activation of Cl(-) secretion, which was completely inhibited by 293B, a specific inhibitor of cAMP-stimulated basolateral KCNQ1/KCNE3 K(+) channels. We therefore speculated that a basolateral P2Y(6) receptor could increase cAMP. Indeed UDP elevated cAMP in isolated crypts. We identified an epithelial P2Y(6) receptor using crypt [Ca(2+)](i) measurements, RT-PCR, and immunohistochemistry. To investigate whether the rat P2Y(6)elevates cAMP, we coexpressed the P2Y(1) or P2Y(6) receptor together with the cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel in Xenopus oocytes. A two-electrode voltage clamp was used to monitor nucleotide-induced Cl(-) currents. In oocytes expressing the P2Y(1) receptor, ATP transiently activated the endogenous Ca(2+)-activated Cl(-) current, but not CFTR. In contrast, in oocytes expressing the P2Y(6)receptor, UDP transiently activated the Ca(2+)-activated Cl(-) current and subsequently CFTR. CFTR Cl(-) currents were identified by their halide conductance sequence. In summary we find a basolateral P2Y(6) receptor in colonic epithelial cells stimulating sustained NaCl secretion by way of a synergistic increase of [Ca(2+)](i) and cAMP. In support of these data P2Y(6) receptor stimulation differentially activates CFTR in Xenopus oocytes.
The final adjustment of renal sodium and calcium excretion is achieved by the distal nephron, in which transepithelial ion transport is under control of various hormones, tubular fluid composition, and flow rate. Acquired or inherited diseases leading to deranged renal sodium and calcium balance have been linked to dysfunction of the distal nephron. Diuretic drugs elicit their effects on sodium balance by specifically inhibiting sodium transport proteins in the apical plasma membrane of distal nephron segments. The identification of the major apical sodium transport proteins allows study of their precise distribution pattern along the distal nephron and helps address their cellular and molecular regulation under various physiological and pathophysiological settings. This review focuses on the topological arrangement of sodium and calcium transport proteins along the cortical distal nephron and on some aspects of their functional regulation. The availability of data on the distribution of transporters in various species points to the strengths, as well as to the limitations, of animal models for the extrapolation to humans.
The relative importance of plasma membrane Ca2+-ATPase (PMCA) 1 and PMCA4 was assessed in mice carrying null mutations in their genes (Atp2b1 and Atp2b4). Loss of both copies of the gene encoding PMCA1 caused embryolethality, whereas heterozygous mutants had no overt disease phenotype. Despite widespread and abundant expression of PMCA4, PMCA4 null (Pmca4-/-) mutants exhibited no embryolethality and appeared outwardly normal. Loss of PMCA4 impaired phasic contractions and caused apoptosis in portal vein smooth muscle in vitro; however, this phenotype was dependent on the mouse strain being employed. Pmca4-/- mice on a Black Swiss background did not exhibit the phenotype unless they also carried a null mutation in one copy of the Pmca1 gene. Pmca4-/- male mice were infertile but had normal spermatogenesis and mating behavior. Pmca4-/- sperm that had not undergone capacitation exhibited normal motility but could not achieve hyperactivated motility needed to traverse the female genital tract. Ultrastructure of the motility apparatus in Pmca4-/- sperm tails was normal, but an increased incidence of mitochondrial condensation indicated Ca2+ overload. Immunoblotting and immunohistochemistry showed that PMCA4 is the most abundant isoform in testis and sperm and that it is localized to the principle piece of the sperm tail, which is also the location of the major Ca2+ channel (CatSper) required for sperm motility. These results are consistent with an essential housekeeping or developmental function for PMCA1, but not PMCA4, and show that PMCA4 expression in the principle piece of the sperm tail is essential for hyperactivated motility and male fertility.
Plasma membrane Ca(2+) ATPases (PMCAs) are ubiquitous in Ca(2+)-transporting organs, including the kidney. Using RT-PCR, we detected PMCA1b, PMCA2b (rare), and PMCA4b in Madin-Darby canine kidney (MDCK) cells. At the protein level, only PMCA1 and PMCA4 were readily detected and were highly enriched in the basolateral membrane. The Na(+)/Ca(2+) exchanger NCX1 was also detected at the transcript and protein level. A functional assay measuring (45)Ca(2+) flux across MDCK cell monolayers under resting conditions indicated that two-thirds of apicobasolateral Ca(2+) transport was provided by Na(+)/Ca(2+) exchanger and one-third by PMCAs, as determined in Na(+)-free media and using various PMCA inhibitors (La(3+), vanadate, calmidazolium, and trifluoroperazine). The importance of PMCA4b for basolateral Ca(2+) efflux was demonstrated by overexpression of PMCA4b or antisense knockdown of endogenous PMCA4b. Overexpression of PMCA4b increased apicobasolateral Ca(2+) transport to approximately 140%, whereas antisense treatment reduced Ca(2+) flux approximately 45% compared with controls. The MDCK system is thus an ideal model for functional studies of the specific role and regulation of PMCA isoforms in Ca(2+) reabsorption in the distal kidney.
Growth hormone (GH) has antidiuretic and antinatriuretic effects in rats and humans, but the molecular mechanisms responsible for these effects are unknown. The aim of this study was to investigate the mechanisms behind the acute renal effects of GH in rats. Female rats received rat (r)GH (2.8 mg/kg sc) or saline and were placed in metabolic cages for 5 h. Urinary excretion of electrolytes and urinary volume were reduced after rGH injection, while urine osmolality was increased. Creatinine and lithium clearance remained unchanged, suggesting that rGH increases reabsorption in segments distal to the proximal tubule. Total plasma insulin-like growth factor I (IGF-I) levels did not change, while cortical IGF-I mRNA abundance was increased. The relative abundance of total and Ser(256)-phosphorylated aquaporin 2 was found to be unchanged by immunoblotting, whereas a significant increase of Thr(96) and Thr(101)-phosphorylated NKCC2 (renal Na(+), K(+), 2Cl(-) cotransporter) was found in the inner stripe of outer medulla thick ascending limbs (mTAL). Additionally, an increased NKCC2 expression was observed in the cortical region. Immunohistochemistry confirmed these findings. The density of NKCC2 molecules in the apical membrane of mTAL cells appeared to be unchanged after rGH injection evaluated by immunoelectron microscopy. Basolateral addition of rGH or IGF-I to microperfused rat mTAL segments did not change transepithelial voltage. In conclusion, GH appears to exert its acute antinatriuretic and antidiuretic effects through indirect activation of NKCC2 in the mTAL.
Calcium (Ca(2+)) is a key constituent in a myriad of physiological processes from intracellular signalling to the mineralization of bone. As a consequence, Ca(2+) is maintained within narrow limits when circulating in plasma. This is accomplished via regulated interplay between intestinal absorption, renal tubular reabsorption, and exchange with bone. Many studies have focused on the highly regulated active transcellular transport pathways for Ca(2+) from the duodenum of the intestine and the distal nephron of the kidney. However, comparatively little work has examined the molecular constituents creating the paracellular shunt across intestinal and renal epithelium, the transport pathway responsible for the majority of transepithelial Ca(2+) flux. More specifically, passive paracellular Ca(2+) absorption occurs across the majority of the intestine in addition to the renal proximal tubule and thick ascending limb of Henle's loop. Importantly, recent studies demonstrated that Ca(2+) transport through the paracellular shunt is significantly regulated. Therefore, we have summarized the evidence for different modes of paracellular Ca(2+) flux across renal and intestinal epithelia and highlighted recent molecular insights into both the mechanism of secondarily active paracellular Ca(2+) movement and the identity of claudins that permit the passage of Ca(2+) through the tight junction of these epithelia.
Fine-tuning of renal calcium ion (Ca(2+)) reabsorption takes place in the distal convoluted and connecting tubules (distal convolution) of the kidney via transcellular Ca(2+) transport, a process controlled by the epithelial Ca(2+) channel Transient Receptor Potential Vanilloid 5 (TRPV5). Studies to delineate the molecular mechanism of transcellular Ca(2+) transport are seriously hampered by the lack of a suitable cell model. The present study describes the establishment and validation of a primary murine cell model of the distal convolution. Viable kidney tubules were isolated from mice expressing enhanced Green Fluorescent Protein (eGFP) under the control of a TRPV5 promoter (pTRPV5-eGFP), using Complex Object Parametric Analyser and Sorting (COPAS) technology. Tubules were grown into tight monolayers on semi-permeable supports. Radioactive (45)Ca(2+) assays showed apical-to-basolateral transport rates of 13.5 ± 1.2 nmol/h/cm(2), which were enhanced by the calciotropic hormones parathyroid hormone and 1,25-dihydroxy vitamin D3. Cell cultures lacking TRPV5, generated by crossbreeding pTRPV5-eGFP with TRPV5 knockout mice (TRPV5(-/-)), showed significantly reduced transepithelial Ca(2+) transport (26 % of control), for the first time directly confirming the key role of TRPV5. Most importantly, using this cell model, a novel molecular player in transepithelial Ca(2+) transport was identified: mRNA analysis revealed that ATP-dependent Ca(2+)-ATPase 4 (PMCA4) instead of PMCA1 was enriched in isolated tubules and downregulated in TRPV5(-/-) material. Immunohistochemical stainings confirmed co-localization of PMCA4 with TRPV5 in the distal convolution. In conclusion, a novel primary cell model with TRPV5-dependent Ca(2+) transport characteristics was successfully established, enabling comprehensive studies of transcellular Ca(2+) transport.
Kidney stones are a prevalent clinical condition imposing a large economic burden to the healthcare system. Hypercalciuria remains the major risk factor for development of a Ca(2+)-containing stone. The kidney's ability to alter Ca(2+) excretion in response to changes in serum Ca(2+) is in part mediated by the Ca(2+)-sensing receptor (CaSR). Recent studies revealed renal claudin-14 (Cldn14) expression localized to the thick ascending limb (TAL) and its expression to be regulated via the CaSR. We find that Cldn14 expression is increased by high dietary Ca(2+) intake and by elevated serum Ca(2+) levels induced by prolonged 1,25-dihydroxyvitamin D(3) administration. Consistent with this, activation of the CaSR in vivo via administration of the calcimimetic cinacalcet hydrochloride, led to a 40-fold increase in Cldn14 mRNA. Moreover, overexpression of Cldn14 in two separate cell culture models decreased paracellular Ca(2+) flux by preferentially decreasing cation permeability, thereby increasing transepithelial resistance. These data support the existence of a mechanism whereby activation of the CaSR in the TAL increases Cldn14 expression, which in turn blocks the paracellular reabsorption of Ca(2+). This molecular mechanism likely facilitates renal Ca(2+) losses in response to elevated serum Ca(2+). Moreover, dysregulation of the newly described CaSR-Cldn14 axis likely contributes to the development of hypercalciuria and kidney stones.
Ca2+ and Mg2+ are essential ions in awide variety of cellular processes and form amajor constituent of bone. It is, therefore, essential
that the balance of these ions is strictly maintained. In the last decade, major breakthrough discoveries have vastly expanded
our knowledge of the mechanisms underlying epithelial Ca2+ and Mg2+ transport. The genetic defects underlying various disorders with altered Ca2+ and/or Mg2+ handling have been determined. Recently, this yielded the molecular identification of TRPM6 as the gatekeeper of epithelial
Mg2+ transport. Furthermore, expression cloning strategies have elucidated two novel members of the transient receptor potential
family, TRPV5 and TRPV6, as pivotal ion channels determining transcellular Ca2+ transport. These two channels are regulated by avariety of factors, some historically strongly linked to Ca2+ homeostasis, others identified in amore serendipitous manner. Herein we review the processes of epithelial Ca2+ and Mg2+ transport, the molecular mechanisms involved, and the various forms of regulation.
Maintenance of plasma Ca(2+) and Mg(2+) levels is of vital importance for many physiological functions. This is achieved via a coordinated interplay between the intestine, bone and kidney by amending the rate of absorption, storage and excretion, respectively. Discovery of the transient receptor potential (TRP) family identified several new ion channels acting as gatekeepers of Ca(2+) and Mg(2+) transport in these epithelia, greatly increasing our understanding of the molecular processes that facilitate the movement of these minerals. In the intestine, TRP channels contribute to the saturable active transcellular movement of divalent cations from the lumen into the enterocyte. Furthermore, in bone, TRPV channels play important roles by influencing the osteoclastic resorption process, thereby contributing importantly to overall bone mineral content. The divalent cation-permeable TRPV5 and TRPM6 channels are located in the renal distal convolution, the main site of active transcellular Ca(2+) and Mg(2+) transport. The channels are regulated by a multitude of factors and hormones that contribute importantly to keeping the systemic concentrations of Ca(2+) and Mg(2+) within normal limits. Dysregulation of either channel impacts the renal reabsorptive capacity for these cations. This review summarizes the current knowledge related to TRP channels in epithelial Ca(2+) and Mg(2+) transport.
To identify the forms of the plasma membrane calcium pump present in tissues that transport calcium, cDNA from human placenta and proximal small intestine was amplified by the polymerase chain reaction using a pair of mixed primers based on all the known human and rat plasma membrane calcium pump sequences. Clones were identified from the two human forms HPMCA1 and HPMCA4, but no new sequences were found in either tissue. RNA blots probed with HPMCA1 showed two bands in both tissues; probing with HPMCA4 gave a single, larger species. In placenta, HPMCA4 was the more abundant form and similar expression was found in full-term and second-trimester placentas. In contrast, in the small intestine, HPMCA1 was more abundant, suggesting that calcium absorption is not associated with any one specific isoform in calcium transporting cells.
We have studied localization of mRNAs coding isozymes of rat plasma membrane Ca(2+)-adenosinetriphosphatase pump (rPMCA) in the rat kidney, with use of reverse transcription (RT) with subsequent amplification by polymerase chain reaction (PCR). When zones of the kidney were separated by macrodissection, a large amount of mRNA coding isozyme rPMCA1 was found in all zones; mRNA for isozyme rPMCA2 was abundant in cortex and in outer medulla, and mRNA for isozyme rPMCA3 was prominent in outer medulla. The mRNAs were analyzed in microdissected cortical nephron segments by use of RT-PCR approach described previously [T. Moriyama, H. R. Murphy, B. M. Martin, and A. Garcia-Perez. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F1470-F1474, 1990]. We detected mRNA for isozyme rPMCA2 in microdissected distal convoluted tubules (DCT) and in cortical thick ascending limbs (CTAL) and, less consistently, also in proximal convoluted tubule and in glomeruli. The mRNA for isozyme rPMCA1 was abundant in glomeruli but was absent in all examined cortical tubular segments. Our results document that mRNAs for all three major isozymes of rPMCA are present and show a unique distribution in the three major zones of rat renal parenchyma. Specific mRNA coding for rPMCA2 was detected in cortical tubules, namely in CTAL and DCT, whereas mRNA coding isozyme rPMCA1 was found in glomeruli. We suggest that isozyme rPMCA2 might be specifically related to epithelial cells and their function, whereas rPMCA1 is probably a component of nonepithelial cells including these in glomeruli.
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+ (Na
b+). Isoosmotic substitution of Na
b+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 Ca
b2+resulted in a significant increase in Ca2+ transport both in the presence and absence of Na
b+. 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.
Distal nephron segments are heterogenous with respect to adenylate cyclase responses to stimulation with parathyroid hormone (PTH) or calcitonin (CT). We examined effects of these hormones and of 8-(p-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPTcAMP) on net Ca absorption (Jnet Ca2+, pmol.min-1.mm-1) in rabbit distal nephron segments by in vitro microperfusion technique. We studied three segments, including distal convoluted tubule (DCT), connecting tubule (CNT), and cortical collecting duct (CCD). PTH (1 nM) in bath significantly increased Jnet Ca2+ from 2.28 +/- 0.35 to 9.44 +/- 1.13 in CNT, but did not affect Jnet Ca2+ in DCT or CCD. CT (1 nM) in bath significantly increased Jnet Ca2+ from 1.58 +/- 0.29 to 4.45 +/- 1.01 in DCT, whereas it did not affect Jnet Ca2+ either in CNT or in CCD. CPTcAMP (30 microM) in bath significantly increased Jnet Ca2+ from 2.29 +/- 0.42 to 3.97 +/- 0.43 in DCT and from 2.43 +/- 0.18 to 5.83 +/- 0.37 in CNT, but it did not affect Jnet Ca2+ in CCD. When Na+ was removed from bathing fluid or when 0.1 mM ouabain was added to bath, Jnet Ca2+ in both DCT and CNT significantly decreased. Furthermore, stimulatory effects of PTH and CT on Ca2+ absorption in the respective segments were abolished under these conditions. These results suggest that PTH and CT increase Ca2+ absorption in CNT and DCT, respectively, through cAMP-mediated mechanisms. Presence of a basolateral Na(+)-Ca2+ exchange process seems to be a prerequisite for effects of these hormones. However, exact intracellular mechanisms remain uncertain.
We used a monoclonal antibody (5F10) specific for the human erythrocyte plasma membrane Ca(++)-pump to demonstrate the presence and distribution of Ca(++)-pump epitopes in rat intestine. In paraffin embedded tissue sections, antibody 5F10 binds to epitopes in the basolateral membranes of absorptive cells in rat duodenum and portions of jejunum but not ileum. Western blot analysis of intestinal mucosal proteins with antibody 5F10 shows binding of antibody to major bands of Mr approximately 135,000 and Mr approximately 72,000, and to lesser bands of Mr approximately 125,000 and Mr approximately 27,000. This pattern was seen in mucosal homogenates of rat duodenal and jejunal cells and to a lesser extent in ileal cells. The Mr approximately 135,000 band corresponds to the molecular weight of Ca(++)-pumps in other tissues. The other bands correspond in size to known proteolytic fragments of the Ca(++)-pump. Slot-blot analysis of nitrocellulose immobilized mucosal homogenates shows binding of 5F10 to be greatest in duodenum and least in ileum. Ca(++)-transport studies by the everted gut sac technique show a correlation between vitamin D induction of active Ca(++)-transport and the segmental distribution of Ca(++)-pump epitopes.
A major unanswered question in central nervous system physiology concerns the mechanism by which cerebrospinal fluid (CSF) Ca2+ homeostasis is maintained in the face of hypo- or hypercalcemia. To address this question, we sought and found a protein of Mr approximately 140,000 in choroid plexus plasma membranes that forms a phosphorylated intermediate with characteristics of a plasma membrane Ca2+-pump. A choroid plexus plasma membrane protein of this molecular weight also bound to a monoclonal antibody prepared against the human erythrocyte plasma membrane Ca2+-Mg2+ ATPase Ca2+-pump. When this monoclonal antibody was used for immunohistochemical localization, the plasma membrane Ca2+-pump was found primarily in the CSF-facing membranes of choroid plexus cells from rats, cats, and man. The localization of a plasma membrane Ca2+-pump in the CSF-facing membranes of the choroid plexus suggests that the choroid plexus, by mechanisms including this pump, may regulate CSF Ca2+ concentrations.
In an effort to extend our studies on Ca2+ pumps to animal models, we developed a new monoclonal antibody (5F10) prepared against the human erythrocyte Ca2+-Mg2+-adenosinetriphosphatase (ATPase) that recognizes a protein of approximately 140 kDa in rat kidney homogenates. Enzyme-linked immunosorbent assays show that monoclonal antibody 5F10 binds purified Ca2+-Mg2+-ATPase and rat kidney membrane extracts in a concentration-dependent manner. In paraffin-embedded tissue sections, antibody 5F10 binds to an epitope in the basolateral membranes of rat kidney distal convoluted tubule principal cells. The antibody does not bind to intercalated cells. The latter cells were characterized by the presence of large amounts of carbonic anhydrase C. Polyclonal antibodies directed against chick intestinal 28-kDa vitamin D-dependent calcium binding protein (28-kDa CaBP) also bind epitopes in distal convoluted tubule cells, connecting tubules, and portions of collecting duct but not intercalated cells. Western blot and 45Ca blot analysis of renal cytosolic proteins showed that the polyclonal 28-kDa CaBP-directed antibody detects a protein which also binds calcium. Western blot analysis with monoclonal antibody 5F10 shows binding to both the authentic purified erythrocyte Ca2+ pump (approximately 138 kDa) and to tryptic fragments of this pump. Antibody JA3, previously used for staining of human kidney tubules, reacts with a different set of tryptic fragments, showing that the two antibodies are directed against different regions or conformational determinants on the pump molecule. We show that Ca2+-Mg2+-ATPase and 28-kDa CaBP are present in the principal cells of the distal convoluted tubule of the rat and are absent in intercalated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
These studies examined transepithelial routes of basal and hormone-activated calcium absorption in thick ascending limbs of Henle's loop perfused in vitro. Single cortical (cTAL) or medullary (mTAL) thick ascending limbs were dissected from kidneys of normal mice. Tubules were perfused at 37 degrees C at pH 7.4 in bicarbonate-buffered media for cTALs and in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions for mTALs. Calcium was determined by electrothermal atomic absorption spectrophotometry. External driving forces for calcium were eliminated by perfusing and bathing tubules with symmetric solutions containing identical calcium concentrations and by abolishing the transepithelial voltage (VT), by adding furosemide or bumetanide to the luminal perfusate. Basal calcium absorption averaged 60 X 10(-12) mol.s-1.cm-2 in cTAL. After addition of 1 U/ml parathyroid hormone (PTH)-(1-34), net calcium absorption increased to 162 X 10(-12) mol.s-1.cm-2 (P less than 0.05) with no change in VT. Similarly, addition of 10(-3) M dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) to external bathing solutions increased net calcium absorption to 201 from 70 X 10(-12) mol.s-1.cm-2 in control conditions (P less than 0.025). These results demonstrate that PTH, acting at least in part through cAMP, activates a cellular pathway for calcium absorption in cTAL. In segments of mTAL, calcium absorption was strictly voltage dependent: when VT was abolished, net calcium absorption fell to values indistinguishable from zero and was not altered in the presence of dibutyryl cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)
The presence of an Na+/Ca2+ exchange system in basolateral plasma membranes from rat small intestinal epithelium has been demonstrated by studying Na+ gradient-dependent Ca2+ uptake and the inhibition of ATP-dependent Ca2+ accumulation by Na+. The presence of 75 mM Na+ in the uptake solution reduces ATP-dependent Ca2+ transport by 45%, despite the fact that Na+ does not affect Ca2+-ATPase activity. Preincubation of the membrane vesicles with ouabain or monensin reduces the Na+ inhibition of ATP-dependent Ca2+ uptake to 20%, apparently by preventing accumulation of Na+ in the vesicles realized by the Na+-pump.l It was concluded that high intravesicular Na+ competes with Ca2+ from intravesicular Ca2+ binding sites. In the presence of ouabain, the inhibition of ATP-dependent Ca2+ transport shows a sigmoidal dependence on the Na+ concentration, suggesting cooperative interaction between counter transport of at least two sodium ions for one calcium ion. The apparent affinity for Na+ is between 15 and 20 mM. Uptake of Ca2+ in the absence of ATP can be enhanced by an Na+ gradient (Na+ inside greater than Na+ outside). This Na+ gradient-dependent Ca2+ uptake is further stimulated by an inside positive membrane potential but abolished by monensin. The apparent affinity for Ca2+ of this system is below 1 microM. In contrast to the ATP-dependent Ca2+ transport, there is no significant difference in Na+ gradient-dependent Ca2+ uptake between basolateral vesicles from duodenum, midjejunum and terminal ileum. In duodenum the activity of ATP-driven Ca2+ uptake is 5-times greater than the greater than the Na+/Ca2+ exchange capacity but in the ileum both systems are of equal potency. Furthermore, the Na+/Ca2+ exchange mechanism is not subject to regulation by 1 alpha, 25-dihydroxy vitamin D-3, since repletion of vitamin D-deficient rats with this seco-steroid hormone does not influence the Na+/Ca2+ exchange system while it doubles the ATP-driven Ca2+ pump activity.
The effects of human calcitonin (HCT) on the distal tubule were investigated by micropuncture in hormone-deprived rats, i.e. in the absence of parathyroid hormone, antidiuretic hormone and glucagon, which might have masked these effects. Two groups of rats were studied: hormone-deprived and hormone-deprive + HCT, infused at 1.0 mU/min X 100 g.b.w. In the urine, HCT markedly reduced Ca and Mg excretion whereas excretion of water, Na and K was not significantly affected. Along the distal tubule, HCT strongly enhanced Na, Cl, Mg, Ca and total solute reabsorption, decreased K secretion but did not alter water or phosphate transport. It is concluded that HCT stimulated Na, Cl, Ca and Mg reabsorption. If, as suggested, HCT also stimulated the reabsorptive component of K transport, the hormone should therefore elicit the same physiological effects in the distal tubule and the thick ascending limb.
Calcium transport was studied in medullary and cortical segments of the thick ascending limb of Henle perfused in vitro. 45Ca was added to the perfusate for measuring lumen-to-bath flux (JlbCa), to the bath for measuring bath-to-lumen flux (JblCa), or to both perfusate and bath for measuring net flux (JnetCa). In the medullary segment JlbCa exceeding JblCa and the efflux:influx coefficient ratio was not different from the value predicted from the observed potential difference (PD). In the cortical segments, however, efflux:influx coefficient ratio was greater than the value predicted from the PD, suggesting that calcium transport in this segment may be active, while it is passive in the medullary segment. Furosemide, which reversibly decreases PD in both cortical and medullary segments, inhibited JlbCa only in the medullary segment. Parathyroid hormone (PTH), on the other hand, had no effect on JnetCa in the medullary segment, but it significantly augmented JnetCa in the cortical segment. These results indicate that calcium transport in the thick ascending limb is heterogeneous. In the medullary segment it is passive, inhibited by furosemide and not influenced by PTH. In the cortical segment, however, calcium transport appears to be active, not inhibited by furosemide and stimulated by PTH.
The intestinal basolateral membrane Ca(2+)-transporting adenosinetriphosphatase is the energy-dependent step in the absorption of dietary Ca2+ by the vitamin D-dependent transcellular pathway. Multiple plasma membrane Ca(2+)-pump isoforms are produced from four genes (PMCA1 to 4) and alternative mRNA splicing. We have studied which isoforms are detectable in adult human and rat gastrointestinal tissues by polymerase chain reaction (PCR) amplification, sequencing, and blotting. PMCA1 was the predominant gene product amplified from human small intestinal mucosa, although a minor additional variant lacking the exon at splice site B was detected, which resembled that described for PMCA4. Of the variants described at site C, only the shortest transcript of PMCA1 was amplified; both previously described forms of PMCA4 were found, particularly in colon where PMCA4 predominated. From rat intestinal cDNA, mixed primer PCR amplified PMCA1 and a novel sequence, the rat PMCA4 homologue, which was expressed in many tissues including small intestinal muscle and colon. However, PMCA1 was overwhelmingly predominant in the mucosa of the small intestine, being most abundant in duodenum. These results suggest the involvement of the Ca(2+)-pump isoform PMCA1b in intestinal Ca2+ absorption.
Evaluations of 1,270 patients with recurrent nephrolithiasis in an outpatient setting were analyzed for the purpose of updating the classification of nephrolithiasis. All but 4% had abnormal urinary biochemistry that placed them into one or more of 20 etiologic categories. A single diagnosis was documented in 41.3% of patients. The remaining 58.7% had more than one diagnosis. Hypercalciuric calcium (Ca) nephrolithiasis, encountered in 60.9% of patients, comprised six variants--absorptive hypercalciuria Type I and II, renal hypercalciuria, primary hyperparathyroidism, and unclassified hypercalciuria (renal phosphate leak and fasting hypercalciuria). Hyperuricosuria Ca nephrolithiasis (HUCN) and gouty diathesis (GD) accounted for 35.8% and 10.0% of patients, respectively. Distinguishing features were hyperuricosuria and normal urinary pH in HUCN, and normal urinary uric acid and low urinary pH (< 5.5) in GD. Hyperoxaluric Ca nephrolithiasis, occurring in 8.1% of patients, was subdivided into enteric, primary, and dietary variants. Hypocitraturic Ca nephrolithiasis affected 28% of patients in its idiopathic variant. Many of these patients' problems were probably dietary in origin, while some could have had incomplete renal tubular acidosis. Hypocitraturia due to renal tubular acidosis or chronic diarrheal syndrome affected only 3.3% of patients. Hypomagnesiuric Ca nephrolithiasis, infection stones, and cystinuria were uncommon, accounting for 6.8%, 5.9%, and 0.9% of patients, respectively. The acquired problem of low urine volume (< 1 L/d) was found in 15.3% of patients. The remaining 3.5% of patients were difficult to classify despite the presence of abnormal urinary biochemistry.
Hypercalcemia is a very common electrolyte abnormality that is most often seen as a mild elevation in the setting of asymptomatic primary hyperparathyroidism. The hypercalcemia is usually less than 11.2 mg/dL and does not call for specific or vigorous approaches to management. However, more marked elevations in the serum calcium are also seen and call for a more specific approach to management. When the serum calcium is only moderately elevated, the therapeutic approach should be tempered by clinical assessments about the presence of symptoms and the underlying etiology. When the serum calcium is markedly elevated, a more targeted therapeutic approach is called for which now is independent of the presence of symptoms and usually of the underlying etiology. Considerations of hypercalcemia in these three different ranges will tailor the therapeutic approach to the clinical setting.
To survey the presence of the four different isoforms of the plasma membrane calcium pump (PMCA) and their alternative splicing variants in the rat kidney, three major zones (cortex, outer medulla, and inner medulla) were macrodissected and probed for the presence of mRNA encoding these isoforms and their variants at the splicing site C by using reverse transcription-polymerase chain reaction (RT-PCR). Both the cortex and the outer medulla showed PMCA 1b, 2b, 3(a and c), and 4b. Semiquantitative comparisons indicated that isoform 2b is more abundant in the cortex than in the outer medulla and conversely, that isoform 3 (a and c) is more abundant in the outer medulla than in the cortex. The inner medulla showed only mRNA for isoforms 1b and 4b. The nephron segments present in the cortex and outer medulla were microdissected and analyzed by RT-PCR. Isoforms 1b, 2b, and 4b were found in all nephron segments but were found more frequently in tubular segments with high rates of Ca2+ reabsorption, suggesting that these isoforms may be involved in transepithelial transport. On the other hand, mRNA encoding isoform 3 (a and c) was most abundant in descending thin limb of Henle but was detected also in glomeruli and cortical thin ascending limb. Its distinct localization suggests that this isoform might have another function, such as in intracellular signalling.
The amount of calcium absorbed in the intestine depends on habitual calcium intake. When intake is low, active transcellular calcium transport in the duodenum is upregulated and a larger proportion of calcium is absorbed by the active process than by the passive paracellular process that prevails in the jejunum and ileum. Bioavailability of the calcium source-digestibility and solubilization-plays a role under conditions of low calcium intake but is relatively unimportant when calcium intakes are high (e.g. >800 mg/d in people). Vitamin D intake is a second factor, as active calcium transport is directly and proportionally dependent on the presence in the intestinal cell of calbindin D9k, the biosynthesis of which is totally vitamin D dependent. Passive absorption in jejunum and ileum is the major absorptive process when calcium intake is adequate or high. Passive calcium absorption is a complicated function of solubility in the distal small intestine, the length of sojourn of the chyme in a given intestinal segment, and the rate of paracellular diffusion from lumen to lymph and blood. Calcium that reaches the large intestine undergoes absorption there by both active and passive processes. Probably no more than 10% of total calcium absorption takes place in the large intestine, whether calcium intake is low or high. Calcium absorption by the large bowel can assume nutritional importance under conditions of significant small bowel resection.
The expression of the CII splice variant of the plasma membrane Ca(2+) ATPase 4 (PMCA4) was down-regulated in granule neurons when they were cultured under conditions of partial membrane depolarization (25 mM KCl), which are required for long term in vitro survival of the neurons. These conditions, which cause a chronic increase of the resting free Ca(2+) concentration in the neurons, have recently been shown to promote up-regulation of the PMCA2, 3, and 1CII isoforms. Whereas the chronic, i.e. >3 days, Ca(2+) increase was necessary for the up-regulation of the PMCA1CII, 2, and 3, the down-regulation of the PMCA4CII mRNA was already evident 1-2 h after the start of culturing in 25 mM KCl. The immunosuppressant calcineurin inhibitor FK506 inhibited the down-regulation of the PMCA4CII at both the protein and the mRNA level but did not affect the changes of the other PMCA pumps. Direct evidence for the involvement of calcineurin in the down-regulation of the PMCA4CII was obtained by overexpressing a truncated, constitutively active, and Ca(2+)-independent form of calcineurin; under these conditions, depolarization was not required for the down-regulation of the PMCA4CII pump. De novo synthesis of (transcription) factors was required for the down-regulation of the PMCA4CII mRNA. Calcineurin, therefore, controls the neuronal transcription of PMCA4CII, a splice variant of the pump isoforms that is found almost exclusively in brain.
The recently cloned epithelial Ca2+ channel (ECaC), which is expressed primarily in 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-responsive Ca2+ -transporting epithelia, is postulated to constitute the rate-limiting step in active Ca2+ reabsorption. In the present study, the effect of 1,25(OH)(2)D(3) was investigated on ECaC mRNA and protein levels in kidneys of rats that were raised on a vitamin D-depleting diet. This diet decreased the serum 1,25(OH)(2)D(3) concentration significantly, which was accompanied by a marked drop in serum Ca2+ level. Both 1,25(OH)(2)D(3) and Ca2+ levels were normalized within 48 h after 1,25(OH)(2)D(3) administration. In 1,25(OH)(2)D(3)-deficient rats, ECaC mRNA and protein levels of the kidney cortex were significantly decreased compared with the repleted animals, suggesting that 1,25(OH)(2)D(3) exerts its stimulatory effect on Ca2+ reabsorption via increased ECaC expression. In agreement with this observation, the elucidated human ECaC promoter contains several consensus vitamin D-responsive elements. ECaC was restricted to the apical membrane of the distal part of the distal convoluted and the connecting tubule. This conclusion was based on only minor overlap with the localization of the thiazide-sensitive NaCl co-transporter and complete co-localization with the 1,25(OH)(2)D(3)-dependent Ca2+ binding protein, calbindin-D(28K). In conclusion, ECaC, present in the distal part of the nephron, is an important target for 1,25(OH)(2)D(3)-mediated Ca2+ reabsorption.
The exact distributions of the different salt transport systems along the human cortical distal nephron are unknown. Immunohistochemistry was performed on serial cryostat sections of healthy parts of tumor nephrectomized human kidneys to study the distributions in the distal convolution of the thiazide-sensitive Na-Cl cotransporter (NCC), the beta subunit of the amiloride-sensitive epithelial Na channel (ENaC), the vasopressin-sensitive water channel aquaporin 2 (AQP2), and aquaporin 3 (AQP3), the H(+) ATPase, the Na-Ca exchanger (NCX), plasma membrane calcium-ATPase, and calbindin-D28k (CaBP). The entire human distal convolution and the cortical collecting duct (CCD) display calbindin-D28k, although in variable amounts. Approximately 30% of the distal convolution profiles reveal NCC, characterizing the distal convoluted tubule. NCC overlaps with ENaC in a short portion at the end of the distal convoluted tubule. ENaC is displayed all along the connecting tubule (70% of the distal convolution) and the CCD. The major part of the connecting tubule and the CCD coexpress aquaporin 2 with ENaC. Intercalated cells, undetected in the first 20% of the distal convolution, were interspersed among the segment-specific cells of the remainder of the distal convolution, and of the CCD. The basolateral calcium extruding proteins, Na-Ca exchanger (NCX), and the plasma membrane Ca(2+)-ATPase were found all along the distal convolution, and, in contrast to other species, along the CCD, although in varying amounts. The knowledge regarding the precise distribution patterns of transport proteins in the human distal nephron and the knowledge regarding the differences from that in laboratory animals may be helpful for diagnostic purposes and may also help refine the therapeutic management of electrolyte disorders.
The epithelial Ca2+ channels TRPV5 and TRPV6 are localized to the brush border membrane of intestinal cells and constitute the postulated rate-limiting entry step of active Ca2+ absorption. The aim of the present study was to investigate the hormonal regulation of these channels. To this end, the effect of 17beta-estradiol (17beta-E2), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and dietary Ca2+ on the expression of the duodenal Ca2+ transport proteins was investigated in vivo and analyzed using realtime quantitative PCR. Supplementation with 17beta-E2 increased duodenal gene expression of TRPV5 and TRPV6 but also calbindin-D9K and plasma membrane Ca2+-ATPase (PMCA1b) in ovariectomized rats. 25-Hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) knockout mice are characterized by hyperparathyroidism, rickets, hypocalcemia, and undetectable levels of 1,25(OH)2D3 and were used to study the 1,25(OH)2D3-dependency of the stimulatory effects of 17beta-E2. Treatment with 17beta-E2 upregulated mRNA levels of duodenal TRPV6 in these 1alpha-OHase knockout mice, which was accompanied by increased serum Ca2+ concentrations from 1.69 +/- 0.10 to 2.03 +/- 0.12 mM (P < 0.05). In addition, high dietary Ca2+ intake normalized serum Ca2+ in these mice and upregulated expression of genes encoding the duodenal Ca2+ transport proteins except for PMCA1b. Supplementation with 1,25(OH)2D3 resulted in increased expression of TRPV6, calbindin-D9K, and PMCA1b and normalization of serum Ca2+. Expression levels of duodenal TRPV5 mRNA are below detection limits in these 1alpha-OHase knockout mice, but supplementation with 1,25(OH)2D3 upregulated the expression to significant levels. In conclusion, TRPV5 and TRPV6 are regulated by 17beta-E2 and 1,25(OH)2D3, whereas dietary Ca2+ is positively involved in the regulation of TRPV6 only.
In vascular smooth muscle cells (SMCs), several mechanisms act in concert to regulate the intracellular calcium concentration [Ca2+]i, which may in turn affect vascular tone. One such mechanism is the extrusion of Ca2+ by the plasma membrane calcium ATPase (PMCA). To address, in particular, the role of the neuronal nitric oxide synthase (nNOS)-associating isoform PMCA4b in regulating vascular tone, a doxycycline-responsive transgene for human PMCA4b was overexpressed in arterial SMCs of mice. Overexpression of hPMCA4b resulted in a 2-fold increase in total aortic PMCA4 protein expression and significant real-time RT-PCR-documented differences in the levels of endogenous mouse PMCA1, PMCA4, SERCA2, and IP3R1 gene expression in arterial SMCs. Whereas no significant difference in basal [Ca2+]i or Ca2+ sensitivity was observed in vascular SMCs or mesenteric arteries, respectively, from hPMCA4b-overexpressing versus control mice, hPMCA4b-overexpressing mice revealed a reduced set-point and increased extent of myogenic response and heightened sensitivity to vasoconstrictors. Treatment of arteries with an nNOS inhibitor resulted in a reduced set-point and increased extent of the myogenic response in control but not hPMCA4b-overexpressing mice. Moreover, aortic SMCs from hPMCA4b-overexpressing mice exhibited reduced levels of cGMP under both basal and phenylephrine-stimulated conditions. These changes were associated with significant doxycycline-reversible elevations in blood pressure. Taken together, these data show that overexpression of hPMCA4b in arterial SMCs increases vascular reactivity and blood pressure, an effect that may be mediated in part by negative regulation of nNOS.
Glucocorticoids, such as prednisolone, are often used in clinic because of their anti-inflammatory and immunosuppressive properties. However, glucocorticoids reduce bone mineral density (BMD) as a side effect. Malabsorption of Ca2+ in the intestine is supposed to play an important role in the etiology of low BMD. To elucidate the mechanism of glucocorticoid-induced Ca2+ malabsorption, the present study investigated the effect of prednisolone on the expression and activity of proteins responsible for active intestinal Ca2+ absorption including the epithelial Ca2+ channel TRPV6, calbindin-D(9K), and the plasma membrane ATPase PMCA1b. Therefore, C57BL/6 mice received 10 mg/kg body wt prednisolone daily by oral gavage for 7 days and were compared with control mice receiving vehicle only. An in vivo 45Ca2+ absorption assay indicated that intestinal Ca2+ absorption was diminished after prednisolone treatment. We showed decreased duodenal TRPV6 and calbindin-D(9K) mRNA and protein abundance in prednisolone-treated compared with control mice, whereas PMCA1b mRNA levels were not altered. Importantly, detailed expression studies demonstrated that in mice these Ca2+ transport proteins are predominantly localized in the first 2 cm of the duodenum. Furthermore, serum Ca2+ and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] concentrations remained unchanged by prednisolone treatment. In conclusion, these data suggest that prednisolone reduces the intestinal Ca2+ absorption capacity through diminished duodenal expression of the active Ca2+ transporters TRPV6 and calbindin-D(9K) independent of systemic 1,25(OH)2D3.