Intensive study on calcium homeostasis regulation over the past several decades has established a systematized construal of its role in living phenomena, leaving us with the impression that this field is fairly defined and understood. However, the unveiling of the molecular function of α-Klotho has recently given new insight into this field. α-Klotho is a unique molecule that plays pivotal roles in: (i) the rapid tuning of extracellular Ca2+ concentration through transepithelial Ca2+ transport; (ii) parathyroid hormone secretion and subsequent Ca2+ increase in the serum, and (iii) the signal transduction of FGF23 that adjusts the calcium concentration by downregulating the production of 1,25(OH)2D3. Through these pathways, α-Klotho participates in the regulation of calcium homeostasis of the CSF and blood/body fluids by its actions in the choroid plexus, parathyroid glands and DCT nephrons. In this regard, α-Klotho is a key player that integrates ‘a multi-step regulatory system of calcium homeostasis’ that rapidly adjusts the extracellular calcium concentration and continuously maintains its concentration within a narrow physiological range.

In Greek mythology, life span is controlled by the three daughters of Zeus and Themis, namely Klotho who combs and spins the thread of life, Lachesis who determines the length of life by measuring the length of thread, and Athropos who cuts the string to bring life to an end. In science, the name Klotho was conferred to a gene that was fortuitously discovered in 1997.

α-klotho ( α-kl) mutant mice were originally described as a short-lived model that displays a variety of premature aging-related phenotypes, such as (i) arteriosclerosis; (ii) ectopic calcification in various soft tissues such as lung, kidney, stomach, heart, and skin; (iii) decreased bone mineral density; (iv) emphysema; (v) uncoordinated movement; (vi) atrophy of the skin, and (vii) severe hyperphosphatemia in association with increased concentrations of 1,25(OH)2D3[1]. The responsible gene for these phenotypes was termed α-kl, and shown to encode a type I membrane protein in an extracellular domain that exhibits significant similarity to β-glycosidases, enzymes involved in the digestion of the sugar moiety of substrates [2].The α-kl gene is predominantly expressed in tissues that are involved in calcium homeostasis; i.e. the parathyroid glands, kidney and the choroid plexus [1, 3]. The parathyroid glands play a key role in systemic calcium homeostasis by monitoring the concentration of extracellular Ca2+ ([Ca2+]e) through the calcium-sensing receptor (CaR) and secreting appropriate levels of parathyroid hormone (PTH) to maintain normal calcium concentrations [4,5,6]. In the kidney, α-Klotho (α-Kl) is exclusively co-expressed with calcium-permeable transient receptor potential V5 (TRPV5) channels, Na+/Ca2+ exchanger-1 (NCX1) and calbindin-D28k (a vitamin D-sensitive intracellular Ca2+-transporting protein) in a specialized region of the nephron segments where transepithelial Ca2+ reabsorption is actively regulated. This co-localization is believed to be important for the homeostatic control of calcium. Indeed, mice lacking TRPV5 display diminished renal Ca2+ reabsorption, which causes severe hypercalciuria and compensatory intestinal uptake of dietary Ca2+[7]. The choroid plexus in many respects functions as ‘the kidney of the brain’ [8]. Its main function is the secretion of cerebrospinal fluid (CSF) that involves the movement of water by osmosis and the unidirectional transport of ions including calcium [9]. The expression of α-Kl in tissues involved in calcium metabolism, led to the prediction that α-Kl might be involved in calcium homeostasis [10].

Calcium homeostasis, an essential system for maintenance of life, is largely regulated by the actions of three major hormones, namely PTH, 1,25(OH)2 vitamin D3, and calcitonin that trigger the response of target cells by binding to their receptors and subsequently control the intake, metabolism, and excretion of calcium. Furthermore, the synthesis and secretion of these hormones are mutually activated or suppressed by each other, and are also controlled by extracellular calcium concentrations monitored by CaRs [6, 11]. In order to clarify the importance of α-Kl, its diverse function in the regulatory network of calcium homeostasis is discussed.

α-Kl has been predicted to be present on the cell surface, as α-kl encodes a type I membrane protein. However, large amounts of α-Kl are detectable in the cytoplasm. In addition, the extracellular domain is cleaved and secreted into blood, CSF and urine [12, 13]. This suggests that α-Kl may have dual actions dependent on its location and form.

Surprisingly, the Na+,K+-ATPase complex (α- and β-subunits) was isolated as binding proteins of two intracellular forms of α-Kl: the 120-kDa premature form, and the 135-kDa mature form. Subcellular fractionation revealed that the complexes of premature α-Kl and Na+,K+-ATPase were found in the endoplasmic reticular (ER) fraction, while mature α-Kl and Na+,K+-ATPase complexes accumulated in the early endosome (EE) and Golgi apparatus/cell membrane fractions. From the cell surface biotinylation analyses, it was also confirmed that although the α1 and β1 subunits of Na+,K+-ATPase were readily detectable both in the cell surface and cytoplasmic fractions, α-Kl was primarily detected not only in the cell surface fraction but also in the cytoplasm. Taken together, this suggests that a subset of Na+,K+-ATPase traffics from the ER to the cell surface in conjunction with α-Kl and α-Kl/Na+,K+-ATPase complexes located in the ER and Glogi apparatus, and abundantly accumulates in the EE fraction including the recycling endosome, ready for recruitment to the cell surface [10] (fig. 1).

Fig. 1

Surface recruitment of Na+,K+-ATPase in correlation with the cleavage and secretion of α-Kl. In α-Kl-expressing cells, Na+,K+-ATPase is recruited to the cell surface by a combination of ‘the conventional pathway’ and ‘the α-Kl-dependent pathway’. Low Ca2+ induces the massive recruitment of Na+,K+-ATPase to the plasma membrane. In contrast, high Ca2+ leads to the decline of such additional recruitment. Accordingly, [Ca2+]e regulates this additional recruitment of Na+,K+-ATPase to the plasma membrane in correlation with the cleavage and secretion of α-Kl.

Fig. 1

Surface recruitment of Na+,K+-ATPase in correlation with the cleavage and secretion of α-Kl. In α-Kl-expressing cells, Na+,K+-ATPase is recruited to the cell surface by a combination of ‘the conventional pathway’ and ‘the α-Kl-dependent pathway’. Low Ca2+ induces the massive recruitment of Na+,K+-ATPase to the plasma membrane. In contrast, high Ca2+ leads to the decline of such additional recruitment. Accordingly, [Ca2+]e regulates this additional recruitment of Na+,K+-ATPase to the plasma membrane in correlation with the cleavage and secretion of α-Kl.

Close modal

Na+,K+-ATPase is an ubiquitously expressed housekeeping membrane-bound pump that transports two K+ in and three Na+ out of the cell [14]. Although the expression level of Na+,K+-ATPase varies among tissues, it is significantly high in the kidney DCT cells, choroid plexus and the parathyroid glands, suggesting that highly expressed Na+,K+-ATPase in these α-Kl-expressing cells plays a specific role in cooperation with α-Kl. Notably, electrochemical gradients created by Na+,K+-ATPase reconcile the activities of other ATPases, ion exchanger and ion channels, and influence physiological events occurring around the cell membrane, e.g. the secretion of hormones [15].

The interaction of α-Kl and Na+,K+-ATPase raised an intriguing hypothesis that α-Kl directly affects Na+,K+-ATPase activity. Based on the facts that (i) the α-Kl and Na+,K+-ATPase complexes are preferentially localized intracellularly, and (ii) the catalytic action of Na+,K+-ATPase is an event that is regulated on the cell surface, the regulation of catalytic efficiency by α-Kl was ruled out as unlikely, and instead focus was put on its regulatory role in the recruitment of Na+,K+-ATPase to the cell surface.

Interestingly, the activity of Na+,K+-ATPase in the choroid plexus is inversely correlated with the [Ca2+]e. When incubated in a low Ca2+ solution, the activity of Na+,K+-ATPase increased rapidly, while decreasing in a high Ca2+ solution. Consistent with this observation, low Ca2+ levels in media significantly increased the surface amount of Na+,K+-ATPase and conversely high Ca2+ levels decreased it. Notably, the fluctuation of the amount of Na+,K+-ATPase at the epithelial cell surface rapidly changes and it becomes detectable within 30 s after the shift of [Ca2+]e. This result demonstrates that Na+,K+-ATPase activity is correlated with its amount on the cell surface, both of which respond to [Ca2+]e concentrations, leading us to conclude that the shift of [Ca2+]e triggers a rapid response that recruits Na+,K+-ATPase to the plasma membrane. As expected, α-Kl is required for the rapid recruitment of Na+,K+-ATPase to the cell surface in response to [Ca2+]e concentrations. Furthermore, secretion of α-Kl is induced in response to a low [Ca2+]e in the kidney, parathyroid glands and the choroid plexus suggesting that [Ca2+]e mediates cleavage of α-Kl in conjunction with the rapid response that recruits Na+,K+-ATPase to the cell surface.

As previously reported, the surface expression of Na+,K+-ATPase is controlled by the balance of recruitment to the plasma membrane and internalization of Na+,K+-ATPase (the conventional pathway) [16]. In α-Kl-expressing cells, Na+,K+-ATPase is recruited to the cell surface by a combination of ‘the conventional pathway’ and ‘the α-Kl-dependent pathway’ (fig. 1). The latter is characterized by Ca2+ dependency and a rapid response. The recruitment to the plasma membrane of Na+,K+-ATPase in α-kl–/– mice is solely dominated by the conventional pathway and probably represents ‘basal’ recruitment. Under normocalcemic conditions, a certain amount of Na+,K+-ATPase is additionally recruited by the α-Kl-dependent pathway. Therefore, the amount/activity of surface Na+,K+-ATPase in wild-type (WT) mice is significantly higher than that of α-kl–/– mice. A low [Ca2+]e induces further recruitment of Na+,K+-ATPase to the plasma membrane. In contrast, high Ca2+ leads to the decline of such additional recruitment. Accordingly, [Ca2+]e regulates this additional recruitment of Na+,K+-ATPase to the plasma membrane in correlation with the cleavage and secretion of α-Kl (fig. 1).

α-Kl was shown to be involved in the regulation of calcium homeostasis through the recruitment of Na+,K+-ATPase to the cell surface in response to [Ca2+]e concentrations [10]. In the choroid plexus, Na+,K+-ATPase is quickly recruited to the plasma membrane in response to [Ca2+]e concentrations. Furthermore, fluctuations in cell surface Na+,K+-ATPase correlate with changes in CSF Ca2+ concentrations. In fact, transcellular transport of calcium in the choroid plexus is impaired in α-kl–/– mice, resulting in a decreased calcium concentration in the CSF [10]. Since it is well known that the Na+ gradient, created by Na+,K+-ATPase, reconciles the activities of other ATPases, ion exchanger and ion channels [17], it can be postulated that the increased Na+ gradient drives the transepithelial transport of Ca2+ in cooperation with NCX-1 which is highly expressed in the apical membrane of the epithelium (fig. 2A).

Fig. 2

Roles of α-Kl in calcium homeostasis. α-Kl binds to Na+,K+-ATPase and regulates the recruitment of Na+,K+-ATPase to the cell surface membrane in response to lowered concentrations of extracellular calcium. α-Kl controls calcium re-absorption in the kidney DCT cells, calcium transportation across the choroid plexus into CSF, and PTH secretion in the parathyroid glands. On the contrary, α-Kl is involved in the signal transduction of Fgf23 that suppresses the gene expression of 1α-hydroxylase in the DCT nephrons that leads to negative regulation of 1,25(OH)2D3 synthesis. Calcium metabolism is governed by complicated reciprocal actions and feedback mechanisms and thus calcium concentrations in serum, body fluids and CSF are maintained within strictly narrow ranges.

Fig. 2

Roles of α-Kl in calcium homeostasis. α-Kl binds to Na+,K+-ATPase and regulates the recruitment of Na+,K+-ATPase to the cell surface membrane in response to lowered concentrations of extracellular calcium. α-Kl controls calcium re-absorption in the kidney DCT cells, calcium transportation across the choroid plexus into CSF, and PTH secretion in the parathyroid glands. On the contrary, α-Kl is involved in the signal transduction of Fgf23 that suppresses the gene expression of 1α-hydroxylase in the DCT nephrons that leads to negative regulation of 1,25(OH)2D3 synthesis. Calcium metabolism is governed by complicated reciprocal actions and feedback mechanisms and thus calcium concentrations in serum, body fluids and CSF are maintained within strictly narrow ranges.

Close modal

Similarly, the responsive increase in surface Na+,K+-ATPase expression may play a pivotal role in Ca2+ reabsorption in kidney DCT cells. The calcium transport across various nephron segments was actively examined in the second half of the last century. In 1963, Lassiter et al. [18] hypothesized that the ascending limb of Henle may be a major site of Ca2+ and N+ reabsorption. Rocha et al. [19] demonstrated net Ca2+ absorption in the thick ascending limb perfused in vitro and concluded that Ca2+ movement in this segment was not an entirely passive one. The renal handling of calcium was then intensively investigated using advanced technologies such as micropuncture, microperfusion of isolated tubule segments, isolation of tubular cell plasma membrane, and electron microprobe analysis, resulting in the clarification of many aspects of complex renal functions [20]. In parallel, the involvement of Ca2+-ATPase [21] and Ca2+/Na+ antiport [22] in Ca2+ transport was suggested, and Shareghi and Stoner [23] found that Ca2+ absorption in the DCT and the granular portion of the cortical collecting duct was significantly enhanced by PTH. In addition, Quamme [24, 25] and Sutton et al.[ 26] reported the effects of calcitonin and [Ca2+]e levels on magnesium and calcium transport in the nephron segments. In 1993, Brown et al. [4] proposed a molecular basis for the above observations by the discovery of the CaR. CaR senses a high concentration of [Ca2+]e and inhibits tubular reabsorption of calcium when [Ca2+]e is increased. Thus, the serum Ca2+ level is excessively elevated in CaR knockout mice, because renal tubular Ca2+ reabsorption is not suppressed [27] (see, Ca2+ Sensor Machineries, described below). However, the molecular mechanism by which transepithelial Ca2+ transport is actively regulated in response to low Ca2+ stimuli in nephron segments remains to be solved.

Collectively, these studies suggested that Ca2+ reabsorption is enhanced directly in response to low Ca2+ stimuli in Henle’s loop, and perhaps in the distal nephron as well as in a cell autonomous manner, independent of calcium-regulating hormones. Accordingly, in their textbook Wilson et al. [[11], p 1321] predicted the following: ‘Ca2+ reabsorption is enhanced directly by tendency to hypocalcemia, which is detected by calcium-sensing receptors in Henle’s loop (and possibly also in the distal nephron) that control transepithelial calcium movements independent of PTH or 1,25(OH)2D3’. Notably, we should pay attention to the reason why they used the term ‘calcium sensing receptors’ but not CaR. This raised the possibility that there may be additional Ca2+ receptors/sensing mechanism(s) that actively regulate Ca2+ reabsorption in the kidney nephron segments. Although its molecular basis has long remained unknown, the following newly proposed mechanism [10] may be the first satisfactory explanation for the prediction proposed by Wilson et al. [11]. That is, the transepithelial transport of Ca2+ is directly triggered and processed by the increased Na+ gradient in cooperation with TRPV5, calbindin-D28k, and NCX-1, all of which are exclusively co-expressed with α-Kl in the nephron segment responsible for the active and regulated Ca2+ reabsorption (fig. 2A). Indeed, regulated reabsorption of calcium in the kidney is impaired in α-kl–/– mice, resulting in the excess excretion of calcium into urine [28].

In the parathyroid glands, PTH secretion is stimulated by the declines in extracellular Ca2+. Indeed, a substantial decease in the serum Ca2+ concentration corresponded to a marked increase in serum PTH in WT mice. However, the serum PTH response in α-kl–/– mice was significantly lower than that of WT mice, suggesting that α-Kl is essential for the regulated secretion of PTH. This observation was further confirmed by a newly established ex vivo PTH secretion system. In WT samples, a significant induction of PTH secretion is detected in response to a low Ca2+ concentration, while complete inhibition is observed in normal and high Ca2+ concentrations. Despite the clear induction of PTH secretion by low Ca2+ in specimens prepared from α-kl–/– mice, its extent was only one fourth of that observed in WT samples which is consistent with the results of the in vivo assay [10].

As reported by Brown et al. [29], the secretion of PTH is dependent on an electrochemical gradient of the plasma membrane created by cell surface Na+,K+-ATPase. In fact, ouabain, a specific inhibitor of Na+,K+-ATPase, inhibited the secretion of PTH by dispersed cells from bovine parathyroid glands, which clearly demonstrated the involvement of Na+,K+-ATPase in this process [29]. To investigate the role of Na+,K+-ATPase in the regulated secretion of PTH, the effect of ouabain was examined using an ex vivo assay system. The extent of PTH release in WT samples treated with ouabain is intriguingly similar to that seen in specimens from α-kl–/– mice without ouabain. Furthermore, ouabain treatment induced no additional inhibitory effects on PTH secretion in α-kl–/– cells, suggesting that, in the PTH-secreting system, α-kl deficiency is equivalent to the inhibition of Na+,K+-ATPase by ouabain (fig. 2A).

Since Brown et al. [29] first suggested the importance of Na+,K+-ATPase in regulating the secretion of PTH, the crucial question of how Na+,K+-ATPase activity is regulated in response to low Ca2+ stimuli in the parathyroid glands has remained open. The identification of α-Kl as a key regulator for the surface recruitment of Na+,K+-ATPase in response to low Ca2+ stimuli offers an answer (fig. 2A). The next question is why the secretion of α-Kl is associated with the recruitment of Na+,K+-ATPase and whether this secreted α-Kl is functional.

As described previously [30], α-Kl regulates the production of 1,25(OH)2D3, a major hormonal determinant of intestinal calcium absorption, by negatively regulating the expression of 1 α–hydroxylase which encodes a rate-limiting enzyme of active vitamin D synthesis. As mice lacking either α-kl or Fgf23 display quite similar phenotypes, these molecules are presumed to be major players in the regulation of a common signaling pathway that controls calcium/phosphorus ion homeostasis [1, 31, 32]. Indeed, α-Kl, in combination with Fgf23, regulates the production of 1,25(OH)2D3 in the kidney. In this pathway, α-Kl is assumed to be necessary for the recognition of Fgf23 by target cells, as Urakawa et al. [33] reported that α-Kl binds to Fgf23 and α-Kl converts the canonical FGF receptor 1c (FgfR1c) to a receptor specific for Fgf23. This enables the high-affinity binding of Fgf23 to the cell surface of the distal convoluted tubule where α-Kl is expressed. Subsequently, α-Kl enhances the ability of Fgf23 to induce phosphorylation of the FGF receptor substrate and extracellular signal-regulated kinase in various types of cells where exogenous α-Kl expression is induced. In summary, the interaction among α-Kl, FGFR, and Fgf23 may be a new type of receptor modulation for signal transduction [33]. As for the regulation of 1 α-hydroxylase gene expression, it is reasonable to speculate that either (i) one or more signal mediators from the distal to the proximal tubule would be required or (ii) some paracrine action of secreted α-Kl would be necessary for this signal transduction to occur, because the 1 α-hydroxylase gene is preferentially expressed in proximal convoluted tubule cells, but not in distal convoluted tubule cells where α-Kl/Fgf23 signal is transduced.

α-Kl can act as a β-glucosidase because of its high similarity with the β-glucosidase family. However, this has been questioned because α-Kl lacks glutamic acid residues that are responsible for the catalytic activity of this enzyme family. Nonetheless, the enzymatic activity of α-Kl is demonstrated. The chimeric α-Kl-human IgG1 Fc protein hydrolyzes the 4-methylumbelliferyl β-D-glucuronide among a series of putative substrates and this enzymatic activity is reduced by addition of the specific inhibitors of β-glucuronidase [34]. In addition to naturally occurring β-glucuronides such as β-estradiol 3-β-D-glucuronide, estrone 3-β-D-glucuronide, and estriol 3-β-D-gluconide, α-Kl hydrolyzes extracellular sugar residues of TRPV5 [13]. Taken together, these data suggest that α-Kl functions as a novel β-glucuronidase, and that steroid β-glucuronides and calcium channel TRPV5 are potential candidates for α-Kl actions.

As described, TRPV5 is exclusively co-expressed with calbindin-D28k, NCX-1 and α-Kl in the DCT epithelial cells responsible for the active and regulated Ca2+ re-absorption in the kidney. Ca2+ enters into the cell at the luminal membrane via the epithelial Ca2+ channel TRPV5 and/or TRPV6 and is sequestered by calbindin-D28k or -D9k. Then, bound Ca2+ diffuses to the basolateral cell surface where Ca2+ is extruded into the blood compartment via NCX1 and/or PMCA1b (fig. 2A). The DCT epithelial cells are well equipped to transport and sustain high rates of Ca2+ influx. This high Ca2+ influx may be obtained by prolonged channel durability at the cell surface before inactivation or internalization, because, regardless of [Ca]e, both TRPV5 and TRPV6 are constitutively active as cation channels. Thus, a key component to create such elevated Ca2+ influx is to increase the amount of TRPV5 and TRPV6 channel expression at the luminal cell surface. The question is how the TRPV5 and TRPV6 channel abundance is controlled. Since a large subset of TRPV5 is located in or near the apical (luminal side) membrane of DCT cells, it is hypothesized that these channels are shuttled from intracellular vesicles into the plasma membrane. This led us to focus on the regulatory mechanisms of intracellular trafficking, stabilization and internalization of TRPV5 and TRPV6. Chang et al. [13] reported a novel mechanism that regulates the abundance of TRPV5 at the luminal cell surface. That is, α-Kl in urine, as a β-glucuronidase, increases TRPV5 channel abundance at the luminal cell surface by hydrolyzing the N-linked extracellular sugar residues of TRPV5. Importantly, the trimming of sugar moieties from TRPV5 induces a significant increase in the plasma membrane localization of TRPV5, but does not affect the Ca2+ uptake activity of the TRPV5 channel. This maintains durable calcium channel activity and membrane calcium permeability in the kidney, resulting in an increased Ca2+ influx from the lumen to preserve normal blood Ca2+ levels by a reduction in Ca2+ loss via urine (fig. 2A). Although the report of Chang et al. [13] illustrates the importance of α-Kl in controlling channel abundance at the cell surface, several questions remain to be addressed. First, Ca2+ reabsorption in the kidney should be regulated in a rapid and dynamic manner. However, the reported hydrolyzing reaction of TRPV5 by α-Kl seems to be very slow. Is the enzyme reaction of α-Kl responsible for such rapid regulation? Second, Ca2+ influx should be tightly linked to the fluctuation of [Ca2+]e. Is it possible to control the enzyme activity of α-Kl in urine in response to the serum Ca2+ concentration? Third, how does α-Kl specifically recognize the sugar moiety of TRPV5 and hydrolyze it among the numerous glycosylated membrane proteins on the luminal cell surface membrane? Fourth, TRPV5 is irreversibly modified. Subsequently, is modified TRPV5 recycled or degraded? How are modified and unmodified TRPV5 proteins differentially recognized?

The fluctuation in [Ca2+]e is sensed by cell surface Ca2+ sensor machineries and subsequently acts on PTH secretion, renal calcium reabsorption, secretion of calcitonin by the thyroid C cells, and probably many other [Ca2+]e-responsive cellular phenomena, resulting in the regulation of extracellular calcium homeostasis. The CaR, a member of G-protein-coupled receptor family identified from bovine parathyroid glands [4], is expressed not only in parathyroid glands, but also in the cerebral cortex, cerebellum, renal cortex and medulla, thyroid gland, testis, lung, ileum, large intestine, and adrenal gland [35,36,37,38]. When parathyroid CaR is activated in response to the increase in [Ca2+]e, inositol-1,4,5-triphosphate accumulates and intracellular calcium rises because of the release of calcium from intracellular stores and the opening of plasma membrane calcium channels. This increase in the intracellular calcium subsequently leads to a decrease in PTH secretion [5]. The pattern of these responses is typical to those mediated through a G protein-coupled receptor (GPCR) and sensitive to the elevation in [Ca2+]e. However, the mechanism that actively induces PTH secretion when extracellular Ca2+ is lowered remains to be fully understood.

As for the transcellular Ca2+ transport in the DCT nephron and choroid plexus, the involvement of CaR seems to be limited or unlikely because the major site of intrarenal CaR expression is in the thick ascending limb of Henle’s loop but not in DCT cells [36], and because CaR is not detectable in the choroid plexus [10]. Furthermore, CaR is known to preferentially respond to increasing Ca2+ concentrations and thus the serum Ca2+ level is excessively elevated in CaR knockout mice, because renal tubular Ca2+ reabsorption is not suppressed in knockout mice [27]. In this context, CaR senses a high concentration of Ca2+ and inhibits tubular reabsorption and PTH secretion. On the other hand, α-Kl and Na+,K+-ATPase are involved in the response to a wide range of Ca2+ concentrations. Transepithelial Ca2+ transport in the DCT nephron and choroid plexus is efficiently induced in response to low [Ca2+]e concentrations and suppressed when Ca2+ concentrations are increased. This indicates that the α-Kl/Na+,K+-ATPase system regulates Ca2+ transport and PTH secretion, at least in part, in a CaR-independent manner. Taken together, these all suggest the importance of a calcium sensor machinery that is distinct from CaR. If so, what kinds of molecules should we focus on to identify Ca2+-sensor molecules? The GPCR family to which CaR belongs is one of the potential targets. Certain types of calcium channels and calcium-permeable TRP channels are also possible candidates.

As shown in figure 2B, calcium homeostasis is maintained by a ‘multi-step response system’ categorized according to the time course into (i) seconds to minutes, (ii) minutes to hours, and (iii) hours to day(s) order regulations. In response to hypocalcemic stimuli, transepithelial Ca2+ transport in the choroid plexus and the α-Kl-expressing nephron segments, and PTH secretion in the parathyroid glands are all triggered by electrochemical gradients created by Na+,K+-ATPase (fig. 2B-I) [10]. Because these responses begin immediately after a decline in Ca2+ concentration and persist for a short time, they can be placed into the ‘seconds to minutes order regulation’ (fig. 2B-I). The PTH-mediated increase in Ca2+, such as Ca2+ reabsorption in the kidney and Ca2+ resorption in the bone, continues for hours [5] and thus belongs to the ‘minutes to hours order regulation’ (fig. 2B-II). The production of 1,25(OH)2D3 and subsequent 1,25(OH)2D3-mediated intestinal calcium uptake are in the order of ‘hours to day(s) regulation’ (fig. 2B-III). In addition, 1,25(OH)2D3 enhances the expression and function of TRPV5 present on the apical membrane of DCT cells, and thereby upregulates Ca2+ reabsorption in the kidney [7, 13]. In turn, increased calcium suppresses the transepithelial Ca2+ transport and PTH secretion. The increased 1,25(OH)2D3 suppresses 1 α-hydroxylase gene expression in two ways; namely a self-negative feedback pathway and an α-Kl/FGF23-mediated signal transduction pathway. Taken together, calcium metabolism is governed by complicated reciprocal actions along with feedback mechanisms in the time course from seconds to minutes, hours and day(s) order regulations. Thus, the extracellular calcium concentration is rapidly adjusted and continuously maintained within strictly narrow ranges.

In order to clarify the importance of α-Kl in the regulation of calcium homeostasis, a global image of α-Kl function in the regulatory network of calcium homeostasis is illustrated in figure 2. α-Kl is involved in ‘seconds to minutes order regulation’ of transepithelial Ca2+ transport and secretion of PTH. Subsequently, α-Kl participates in ‘minutes to hours order regulation’ and ‘hours to day(s) order regulation’ through the action of secreted PTH and PTH-mediated production of 1,25(OH)2D3, respectively. α-Kl also participates in the signal transduction of Fgf23 to adjust the calcium concentration by downregulating the production of 1,25(OH)2D3. Furthermore, α-Kl in urine increases TRPV5 channel abundance at the luminal cell surface by hydrolyzing the sugar residues of TRPV5, which also increase Ca2+ reabsorption in the kidney. Therefore, α-Kl is the key player that integrates a ‘multi-step calcium control system’. Thus it is distinct from the known regulatory molecules (PTH, 1,25(OH)2D3 and CT) and from the direct handling molecules for transepithelial calcium transport such as TRPV5, calbindin-D28k, and NCX-1.

It remains an open question as to why α-kl deficiency results in a multitude of premature aging-related phenotypes. Similar premature aging phenotypes are observed in an independently established line, namely the Fgf23 knockout mice. Fgf23 knockout mice develop severe hyperphosphatemia with increased 1,25(OH)2D3 levels [32]. The overproduction of 1,25(OH)2D3 and altered mineral-ion homeostasis are the major cause of these premature aging-like phenotypes observed in α-kl–/– mice, because the lowering of 1,25(OH)2D3 activity by (i) dietary restriction (a regimen in which α-kl–/– mice are fed a vitamin D-deficient diet) [30], (ii) genetic ablation of 1α-hydroxylase in α-kl–/– mice (unpublished data) or in Fgf23–/– mice [39, 40], or (iii) genetic ablation of the VDR gene in α-kl–/– mice (unpublished data) are all able to rescue the premature aging-like phenotypes and enable these mice to survive normally without obvious abnormalities. These independently established conclusions consistently indicate that α-Kl is the key regulator of calcium homeostasis and clearly explain the major cause of phenotypes observed in α-kl–/– mice and the reason why α-Kl is expressed in the tissues related to calcium regulation. Furthermore, the reduced blood glucose and insulin concentrations observed in α-kl–/– mice can be strikingly improved in both male and female α-kl–/– mice when they are fed a vitamin D-deficient diet, suggesting that the impaired glucose metabolism in α-kl–/– mice is a secondary effect caused by increased vitamin D activity [30, 41]. Therefore, the function of α-Kl differs from that reported in a previous study, namely that it interferes with insulin or insulin-like growth factor signal transduction [42]. Notably, a patient carrying a homozygous missense mutation in α-kl gene exhibited defects in mineral ion homeostasis with marked hyperphosphatemia, hypercalcemia and subsequent ectopic calcifications in soft tissues, as well as elevations in Fgf23 and 1,25(OH)2D3 serum levels, but normal fasting glucose and insulin concentrations [43]; this study provides a compelling evidence that α-Kl has a minimal, if any, effect on glucose metabolism. Of particular interest, the circulatory levels of Fgf23 are about 2,000-fold higher in α-kl null mice [33] than in WT mice; despite higher circulatory levels of Fgf23 in α-kl mutants, these mice show physical, biochemical and morphological features similar to Fgf23 null mice, but not as Fgf23 transgenic mice [44], suggesting that the widely encountered premature aging-like features and altered mineral ion homeostasis in the α-kl null mice are due to an inability of Fgf23 to exert its bioactivities in the absence of α-Kl. In other words, the premature ageing-like phenotypes in α-kl null mice are the consequence of loss of Fgf23 activity rather than impaired glucose homeostasis [45].

The next question that needs to be resolved is how hypervitaminosis D3 and the subsequently altered mineral-ion balance lead to multiple premature ageing-like phenotypes, as documented in both α-kl and Fgf23 null mice [1, 32, 40]. As a master regulator of phosphate homeostasis[46], Fgf23 has convincingly shown how the Fgf23/α-Kl system coordinately maintains physiologic phosphorus balance, but this needs additional studies.

Since the identification of α-Kl in 1997, the molecular function of α-Kl has been the subject of intense study, but a clear answer has been elusive. However, recent advances that have given rise to marked progress in clarifying α-Kl actions can be summarized as follows. (i) α-Kl binds to Na+,K+-ATPase, and Na+,K+-ATPase is recruited to the plasma membrane by a novel α-Kl-dependent pathway in correlation with cleavage and secretion of α-Kl in response to extracellular Ca2+ fluctuation. (ii) The increased Na+ gradient created by Na+,K+-ATPase activity drives the transepithelial transport of Ca2+ in cooperation with the Na+/Ca2+ exchanger in the choroid plexus and kidney, this is defective in α-kl–/– mice. (iii) The regulated PTH secretion in the parathyroid glands is triggered via recruitment of Na+,K+-ATPase to the cell surface in response to extracellular Ca2+ concentrations. (iv) α-Kl, in combination with Fgf23, regulates the production of 1,25(OH)2D3 in the kidney. In this pathway, α-Kl binds to Fgf23, and α-Kl converts the canonical FGF receptor 1c to a specific receptor for Fgf23, enabling the high-affinity binding of Fgf23 to the cell surface of the distal convoluted tubule where α-Kl is expressed. (v) α-Kl in urine increases TRPV5 channel abundance at the luminal cell surface by hydrolyzing the N-linked extracellular sugar residues of TRPV5, resulting in increased Ca2+ influx from the lumen. These unveiled molecular functions of α-Kl provided answers for several important questions regarding the mechanisms of calcium homeostasis, such as: (i) what is the non-hormonal regulatory system that directly responds to the fluctuation of extracellular Ca2+; (ii) how is Na+,K+-ATPase activity enhanced in response to low calcium stimuli in the parathyroid glands; (iii) what is the exact role of Fgf23 in calcium and phosphorus metabolism; (iv) how is Ca2+ influx through TRPV5 controlled in the DCT nephron, and finally (v) how is calcium homeostasis regulated in CSF.

However, several critical questions still remain to be solved. α-Kl binds to Na+,K+-ATPase [10], Fgf receptors and Fgf23 [33], and α-Kl hydrolyzes the sugar moieties of TRPV5 [13]. Does α-Kl recognize these proteins directly or indirectly? Is there any common mechanism? How can we reconcile such diverse functions of α-Kl? What is the Ca2+ sensor machinery and how can we isolate it? How do hypervitaminosis D3 and the subsequently altered mineral-ion balance lead to the multiple phenotypes? How does the Fgf23/α-Kl system regulate phosphorus homeostasis?

α-Kl regulates calcium homeostasis of the CSF and blood/body fluids by its actions in the choroid plexus, and in the parathyroid glands and DCT nephrons, respectively. In this regard, α-Kl might play a pivotal role in calcium metabolism as a regulator that integrates calcium homeostasis, although this concept requires further verification in light of related findings. α-Kl studies provide a new paradigm for the mechanisms controlling the [Ca2+]e that may change current concepts in calcium homeostasis and give rise to new insights into this field. It is of great importance to better understand the fundamental molecular mechanism(s) underlying the pleiotropic actions of α-Kl for possible therapeutics against aging-associated complications.

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