Scholarly article on topic 'Update on FGF23 and Klotho signaling'

Update on FGF23 and Klotho signaling Academic research paper on "Biological sciences"

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{FGF23 / "Fibroblast growth factor-23" / Klotho / Bone / Kidney / "Parathyroid gland"}

Abstract of research paper on Biological sciences, author of scientific article — Reinhold G. Erben

Abstract Fibroblast growth factor-23 (FGF23) is a bone-derived hormone known to suppress phosphate reabsorption and vitamin D hormone production in the kidney. Klotho was originally discovered as an anti-aging factor, but the functional role of Klotho is still a controversial issue. Three major functions have been proposed, a hormonal function of soluble Klotho, an enzymatic function as glycosidase, and the function as an obligatory co-receptor for FGF23 signaling. The purpose of this review is to highlight the recent advances in the area of FGF23 and Klotho signaling in the kidney, in the parathyroid gland, in the cardiovascular system, in bone, and in the central nervous system. During recent years, major new functions of FGF23 and Klotho have been discovered in these organ systems. Based on these novel findings, FGF23 has emerged as a pleiotropic endocrine and auto-/paracrine factor influencing not only mineral metabolism but also cardiovascular function.

Academic research paper on topic "Update on FGF23 and Klotho signaling"

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Molecular and Cellular Endocrinology

journal homepage: www.elsevier.com/locate/mce

Update on FGF23 and Klotho signaling

Reinhold G. Erben*

University of Veterinary Medicine Vienna, Vienna, Austria

ARTICLE INFO ABSTRACT

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone known to suppress phosphate reabsorption and vitamin D hormone production in the kidney. Klotho was originally discovered as an anti-aging factor, but the functional role of Klotho is still a controversial issue. Three major functions have been proposed, a hormonal function of soluble Klotho, an enzymatic function as glycosidase, and the function as an obligatory co-receptor for FGF23 signaling. The purpose of this review is to highlight the recent advances in the area of FGF23 and Klotho signaling in the kidney, in the parathyroid gland, in the cardiovascular system, in bone, and in the central nervous system. During recent years, major new functions of FGF23 and Klotho have been discovered in these organ systems. Based on these novel findings, FGF23 has emerged as a pleiotropic endocrine and auto-/paracrine factor influencing not only mineral metabolism but also cardiovascular function.

© 2016 The Author. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-

ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Article history: Received 23 December 2015 Received in revised form 1 April 2016 Accepted 9 May 2016 Available online 10 May 2016

Keywords: FGF23

Fibroblast growth factor-23

Klotho

Kidney

Parathyroid gland

1. Introduction

Gain-of-function mutations in fibroblast growth factor-23 (FGF23) were discovered as the genetic cause of autosomal dominant hypophosphatemic rickets in the year 2000 (The ADHR Consortium, 2000). Soon after this discovery it was shown that FGF23 is a phosphaturic hormone, reducing phosphate reabsorption from urine through a downregulation of sodium phosphate co-transporters in renal proximal tubular epithelial cells (Shimada et al., 2001, 2004a, 2005). There is solid evidence from a number of different diseases and disease models that excessive amounts of circulating intact FGF23 lead to renal phosphate wasting as long as kidney function is normal (Martin et al., 2012). FGF23 also down-regulates 1a-hydroxylase expression in renal proximal tubules, thereby suppressing the production of the biologically active vitamin D hormone, 1a,25-dihydroxyvitamin D3 (Shimada et al., 2001, 2004a, 2005).

Osteoblasts and osteocytes probably are the major sources for circulating FGF23 in vivo (Martin et al., 2012). The secretion of FGF23 in bone is stimulated by the vitamin D hormone and by increased extracellular phosphate (Fig. 1), forming a feedback loop

* Institute of Physiology, Pathophysiology and Biophysics, Dept. of Biomedical Sciences, University of Veterinary Medicine Vienna, Veterinärplatz 1,1210 Vienna, Austria.

E-mail address: Reinhold.Erben@vetmeduni.ac.at

between bone and kidney (Juppner, 2011; Martin et al., 2012; Kaneko et al., 2015). In addition, increased extracellular calcium is able to augment FGF23 secretion (Quinn et al., 2013). Because activating mutations in FGF receptor 1 (FGFR1) in patients with osteoglophonic dysplasia can lead to increased FGF23 secretion (White et al., 2005) and ablation of Fgfr1 in bone partially rescues the excessive Fgf23 secretion in Hyp mice (Xiao et al., 2014), FGFR1 signaling appears to be involved in the regulation of FGF23 secretion in osteoblasts and osteocytes. However, the intracellular pathways downstream of FGFR1 that regulate FGF23 transcription are currently unknown. There is also accumulating evidence that iron deficiency (Wolf and White, 2014) and pro-inflammatory stimuli enhance FGF23 secretion from bone (Ito et al., 2015; David et al., 2016; Pathak et al., 2016).

To protect FGF23 from intracellular cleavage by the subtilisin-like proprotein convertase furin during the secretory process, FGF23 needs to be O-glycosylated at threonine178 within the cleavage site by polypeptide N-acetylgalactosaminyltransferase 3 (GalNT3). Because only the intact FGF23 molecule is biologically active, failure of glycosylation in loss-of-function mutations of GalNT3 results in secretion of mostly cleaved FGF23, leading to an FGF23 deficiency-like phenotype in men and mice (Topaz et al., 2004; Kato et al., 2006; Ichikawa et al., 2009). More recently, it was discovered that O-glycosylation of FGF23 needs to be counterbalanced by phosphorylation of serine180 near the glycosylation site by family with sequence similarity 20, member C (FAM20C). Loss of function in Fam20C leads to increased circulating intact

http://dx.doi.org/10.1016/j.mce.2016.05.008

0303-7207/© 2016 The Author. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Fig. 1. Pleiotropic endocrine and auto-/paracrine functions of FGF23. FGF23 is mainly produced in bone by osteoblasts and osteocytes. Bony secretion of FGF23 is stimulated by phosphate, parathyroid hormone (PTH), and by the vitamin D hormone 1 a,25(OH)2D3. FGF23 acts independently on renal proximal and distal tubules in a Klotho dependent fashion. In renal proximal tubules, FGF23 inhibits phosphate re-uptake and expression of 1a-hydroxylase, the rate-limiting enzyme for vitamin D hormone production. In distal tubules, FGF23 increases reabsorption of calcium and sodium which may indirectly contribute to vascular calcification and may put additional strain on the heart by salt and volume retention. Furthermore, FGF23 inhibits PTH secretion in parathyroid glands by a Klotho independent signaling mechanism. Recent evidence also suggests that FGF23 induces hypertrophy by a direct, Klotho independent action on cardiomyocytes. Because cardiac expression of FGF23 is increased after experimental myocardial infarction and in chronic kidney disease-induced left ventricular hypertrophy, the heart may also become a source of circulating FGF23 in these conditions. It has recently been uncovered that FGF23 is an auto-/paracrine inhibitor of bone mineralization by suppressing alkaline phosphatase in a Klotho independent fashion. It is still controversial whether FGF23 has direct effects on blood vessels, or whether the vascular effects of FGF23 are mediated indirectly through renal calcium retention and suppression of vitamin D hormone production which may in turn promote endothelial dysfunction.

Fgf23 and hypophosphatemic rickets (Wang et al., 2012). Therefore, both phosphorylation and glycosylation of FGF23 are physiologically essential processes, and it is currently thought that the balance between phosphorylation and glycosylation determines the relative amounts of intact and cleaved FGF23 secreted by osteo-blasts and osteocytes (Tagliabracci et al., 2014). Collectively, the recent findings in this area underscore that better insight into the regulation of posttranslational processing of FGF23 is of crucial importance for a more complete understanding of FGF23 biology.

High affinity binding of FGF23 to target cells requires a receptor complex consisting of FGF receptors and the transmembrane protein aKlotho (Klotho) (Kurosu et al., 2006; Urakawa et al., 2006; Goetz et al., 2012). FGF receptors are tyrosine kinase receptors, leading to phosphorylation of downstream molecules after activation through ligand binding. There are 4 different FGFRs (FGFR1, 2, 3, and 4), and it is still controversial which FGFRs are responsible for the actions of FGF23 in different cell types. There is very good evidence that FGF23 signals through a FGF receptor-1c/Klotho complex (Urakawa et al., 2006; Goetz et al., 2012), but Klotho may also bind to FGFR3 and 4 (Kurosu et al., 2006).

Klotho is a single pass transmembrane protein that shares sequence homology with family I b-glycosidases (Kuro-o et al., 1997). There is only one mammalian aKlotho gene, but there are three isoforms of Klotho protein, namely the transmembrane form, a shed soluble form, and a truncated soluble form produced by alternative splicing of Klotho mRNA (Xu and Sun, 2015). The extracellular domain of Klotho, consisting of the two type I b-glycosidase domains KL1 and KL2, can be shed from the cell surface by membrane-anchored proteolytic enzymes, and released into the extracellular fluid and subsequently the blood stream (Imura et al., 2007). In addition, a soluble truncated Klotho protein isoform can

be produced by alternative splicing of the Klotho mRNA, lacking exons 4 and 5 in mice and KL2 due to a premature stop codon in man, respectively (Matsumura et al., 1998; Shiraki-Iida et al., 1998). Therefore, both the human and the murine soluble truncated Klo-tho protein isoforms consist of KL1 only. Main sites of Klotho expression are renal proximal and distal tubules, the choroid plexus in the brain, and parathyroid glands (Kuro-o et al., 1997; Shiraki-Iida et al., 1998; Urakawa et al., 2006; Imura et al., 2007; Hu et al., 2010; Andrukhova et al., 2012).

Klotho was originally discovered as an anti-aging factor (Kuro-o et al., 1997). In agreement with this notion, Klotho and Fgf23 deficient mice are characterized by a severe aging-like phenotype associated with runting, premature death, ectopic calcifications, organ atrophy, and osteomalacia (Kuro-o et al., 1997; Shimada et al., 2004b; Sitara et al., 2004). However, ablation of vitamin D signaling, using mice lacking a functioning vitamin D receptor completely rescues the premature aging phenotype in Klothoand Fgf23~h mice (Hesse et al., 2007; Anour et al., 2012; Streicher et al., 2012). Notably, Klotho~/~ and Fgf23~/~ mice produce excessive amounts of 1,25(OH)2D due to the lacking suppressive effect of Fgf23 on renal 1a-hydroxylase activity. The anti-aging function of Klotho was initially thought to be based on an inhibitory role of soluble Klotho for insulin signaling (Kurosu et al., 2005). Indeed, Klotho~/~ and Fgf23~/~ mice are characterized by increased peripheral insulin sensitivity (Kurosu et al., 2005; Hesse et al., 2007), and the phenotype of Klotho~/~ mice can be partially rescued by insulin receptor substrate-1 (IRS1) haploinsufficiency (Kurosu et al., 2005). However, it was later shown that lack of Klotho does not alter glucose homeostasis in Klotho~/~fVDRD/D compound mutant mice (Anour et al., 2012), indicating that the enhanced insulin sensitivity in Klotho~/~ mice is secondary to disturbed mineral

homeostasis and that the ability of endogenous Klotho to inhibit insulin/insulin-like growth factor-1 (IGF1) signaling is dispensable for glucose homeostasis. Therefore, it is currently believed that the premature aging phenotype in Klotho-/- and Fgf23-/- mice is caused by intoxication with the vitamin D hormone, leading to severe hypercalcemia and hyperphosphatemia and subsequent organ damage. Because deletion of the NaPi-2a gene or restriction of dietary phosphate intake also ameliorates the phenotype of Klotho-1- and Fgf23-/- mice in the presence of elevated vitamin D hormone levels and hypercalcemia (Stubbs et al., 2007; Ohnishi et al., 2009; Kuro-o, 2013), it is clear that the actual toxicity is not mediated by increased circulating vitamin D hormone per se, but rather primarily by the vitamin D-induced hyperphosphatemia.

Loss-of-function mutations in KLOTHO (Ichikawa et al., 2007), FGF23 (Araya et al., 2005), or GALNT3 (Topaz et al., 2004) cause tumoral calcinosis in humans, a disease characterized by ectopic and vascular calcifications. Similar to Klotho-/- and Fgf23-/- mice, the ectopic calcifications in patients with tumoral calcinosis are associated with increased 1,25(OH)2D serum levels together with hypercalcemia and hyperphosphatemia. Conversely, a patient with gain of Klotho function showed hypophosphatemia and rickets as a result of renal phosphate wasting, probably due to augmented FGF23 signaling (Brownstein et al., 2008). It remains unexplained in this regard why the latter patient also showed parathyroid hyper-plasia and increased circulating intact FGF23 levels. Although patients with tumoral calcinosis are characterized by similar changes in mineral metabolism compared with Klotho-/- and Fgf23-/- mice, they lack signs of premature aging, corroborating the notion that the primary function of FGF23 and Klotho is the regulation of mineral metabolism (Ichikawa et al., 2007).

The functional role of the Klotho protein is still a controversial issue. Three major functions have been proposed, i) a hormonal function of soluble Klotho, ii) an enzymatic function as glycosidase, and iii) the abovementioned function of membrane-bound Klotho as co-receptor for FGF23 (Xu and Sun, 2015). As described in the organ-specific sections below, a hormonal function of soluble

Klotho has been reported in the kidney, in the heart, in blood vessels, and for inhibition of insulin signaling (Fig. 2). However, attempts to identify the receptor for soluble Klotho have been unsuccessful so far (Xu and Sun, 2015). Although Klotho lacks essential active site glutamic acid residues typical for this family of glycosidases (Tohyama et al., 2004), there is evidence suggesting that soluble Klotho may function as an enzyme. Several studies suggested that soluble Klotho may have the ability to alter the function and abundance of membrane glycoproteins by cleaving terminal sugars from sugar chains through a putative glycosidase activity (Chang et al., 2005; Cha et al., 2008; Hu et al., 2010). As described in more detail below, some of the hormonal and enzymatic functions of Klotho are still a matter of controversy. However, it is now clear beyond any doubt that membrane-bound Klotho functions as a co-receptor for FGF23 in FGF23 target tissues (Kurosu et al., 2006; Urakawa et al., 2006). Endocrine FGFs such as FGF23 have low affinity for FGF receptors. However, co-expression of membrane-bound Klotho turns the ubiquitously expressed FGFR1c into a specific receptor for FGF23 by increasing the affinity of the receptor complex by a factor of about 20 (Urakawa et al., 2006; Goetz et al., 2012).

The purpose of this review is to highlight the recent advances in the area of FGF23 and Klotho biology. During recent years, major new functions of FGF23 and Klotho signaling have been discovered in the kidney, in the heart, in bone, in blood vessels, and in the parathyroid gland (Fig. 1). Collectively, the novel findings described below suggest that FGF23 and Klotho are far more than only factors important for phosphate and vitamin D homeostasis. Rather, FGF23 has emerged as a pleiotropic hormone influencing not only mineral metabolism but also cardiovascular function. In addition, FGF23 has recently been identified as an auto-/paracrine regulator of bone mineralization.

2. Kidney

The kidney is clearly one of the main sites of Klotho and FGF23

Fig. 2. Endocrine and auto-/paracrine functions of soluble Klotho. The kidney is the major source of circulating soluble Klotho (sKlotho). sKlotho may act on muscle and adipose tissue to inhibit insulin signaling. Furthermore, sKlotho has been shown to protect against cardiac hypertrophy and vascular calcification. In the kidney, sKlotho released from distal tubules may inhibit phosphate reabsorption in proximal tubules, and sKlotho has been shown to stimulate calcium reabsorption in distal tubules.

action, and under physiological conditions probably the most important one. This notion is illustrated by the fact that the phenotype of mice with a kidney-specific ablation of Klotho recapitulates the phenotype of global Klotho knockout mice (Lindberg et al., 2014). Moreover, because serum soluble Klotho was reduced by about 80% in kidney-specific Klotho knockout mice (Lindberg et al., 2014), the kidney is also probably the major source of soluble Klotho in the bloodstream under physiological conditions (Fig. 2).

It has long been known that FGF23 is a phosphaturic hormone (Shimada et al., 2001). The FGF23-induced increase in urinary phosphate excretion is based on the suppression of the apical membrane expression of the phosphate transporters NaPi-2a and NaPi-2c in renal proximal tubules (Larsson et al., 2004; Shimada et al., 2004a, 2004c). The presence of sodium phosphate transporters in the apical membrane is necessary for re-uptake of filtered phosphate from the urine into the epithelium. It has recently been shown in mice with a kidney-specific and inducible ablation of NaPi-2c that the phosphaturic action of FGF23 is mainly determined by downregulation of the apical membrane abundance of NaPi-2a, and that the role of NaPi-2c is minor in this context (Myakala et al., 2014). However, loss-of-function mutations in NaPi-2c lead to hypophosphatemia due to renal phosphate wasting and a stimulation of vitamin D hormone production in humans (Bergwitz et al., 2006). Therefore, NaPi-2c has a more important role for phosphate metabolism in humans compared with mice.

The molecular mechanism underlying the phosphaturic action of FGF23 has long remained elusive. Based on in situ hybridization (Kuro-o et al., 1997) and immunohistochemical studies using a rat monoclonal anti-Klotho antibody directed against the human KL1 domain (Li et al., 2004), it was believed that Klotho is mainly expressed in distal renal tubules. Moreover, Farrow et al. (2009) showed in a time course study that the earliest changes in ERK phosphorylation occur in distal tubules after injection of FGF23 in mice. Therefore, it was unclear how FGF23 could act on proximal tubules where phosphate reabsorption takes place. One of the explanatory scenarios hypothesized that FGF23 may make distal tubules secrete an unknown paracrine factor that in turn signals back to the proximal tubule to suppress phosphate reabsorption (Farrow et al., 2010; White and Econs, 2008).

Using a polyclonal rabbit anti-Klotho antibody directed against the short intracellular region of the membrane-bound Klotho isoform, we recently showed that Klotho is not only expressed in the basolateral membrane of distal but also of proximal renal tubules (Andrukhova et al., 2012). Because antibodies directed against the KL1 domain also detect the secreted isoform of Klotho, it is likely that the discrepant findings regarding aKlotho expression in the murine kidney (Li et al., 2004; Andrukhova et al., 2012) may be explained by differences in the anti-Klotho antibodies used. Furthermore, we not only showed the presence of the co-receptor Klotho in proximal renal tubules, but also that FGF23 directly downregulates membrane expression of NaPi-2a in renal proximal tubular epithelium (Fig. 1) by phosphorylation of the scaffolding protein Na+/H+ exchange regulatory cofactor (NHERF)-1 through extracellular signal-regulated kinase 1 and 2 (ERK1/2) and serum/ glucocorticoid-regulated kinase-1 (SGK1) signaling in a Klotho dependent fashion (Andrukhova et al., 2012). Phosphorylation of NHERF-1 leads to internalization and degradation of NaPi-2a (Deliot et al., 2005; Weinman et al., 2007). Hence, similar to the other major phosphaturic hormone, parathyroid hormone (PTH), FGF23 signaling targets NHERF-1 to regulate the apical membrane abundance of NaPi2a in proximal tubular epithelium (Weinman et al., 2011; Andrukhova et al., 2012). Clinical evidence suggests that PTH and FGF23 signaling interact in the regulation of renal phosphate reabsorption. Efficient FGF23 signaling in proximal renal

tubules appears to require certain levels of circulating PTH, because the phosphaturic effect of FGF23 is decreased in patients with hypoparathyroidism (Gupta et al., 2004; Geller et al., 2007; Bhadada et al., 2013).

Although the finding that FGF23 acts directly on proximal tubules does not exclude the possibility that FGF23 may trigger the release of additional paracrine signals from distal tubules, it establishes a logical model of the phosphaturic FGF23 action. Recent evidence from experiments in conditional knockout mice lends strong support to this model, because mice with a specific deletion of Fgfrl in proximal renal tubules show hyperphosphatemia and are resistant to the phosphaturic actions of FGF23 (Han et al., 2016).

Proximal tubular epithelial cells express FGFR1, 3, and 4, but not 2 (Gattineni et al., 2009; Andrukhova et al., 2012). There is firm evidence that FGFR1 isoform FGFR1c interacts with Klotho to form an FGFR1c-Klotho receptor complex (Urakawa et al., 2006; Goetz et al., 2012). However, Klotho may also interact with FGFR3 and 4 (Kurosu et al., 2006). Based on the recent work by Gattineni et al. (2014), the phosphaturic action of FGF23 is mediated through FGFR1 and FGFR4. Ablation of both Fgfr1 and Fgfr4 was necessary to blunt the phosphaturic action of FGF23, and to completely inhibit the FGF23-induced increase in MAPK phosphorylation in whole kidney lysates (Gattineni et al., 2014). Along similar lines, it has been shown by genetic ablation of Fgfr3 and Fgfr4 in Hyp mice, which are characterized by increased endogenous Fgf23 secretion, that the suppressive effect of Fgf23 on renal hydroxylase expression and phosphate reabsorption is mediated through a combination of FGFR1, FGFR3, and FGFR4 signaling (Li et al., 2011). However, because mice with a specific deletion of Fgfr1 in proximal renal tubules are resistant to the phosphaturic actions of FGF23, FGFR1 may have a dominant role in mediating proximal renal tubular FGF23 signaling (Han et al., 2016). It is currently unknown whether the FGF23 signaling mechanisms downstream of the different FGFRs are similar or different in renal proximal tubular epithelium. It is also not known whether the FGF23-induced signaling mechanisms involved in the suppression of NaPi-2a membrane abundance and in the suppression of renal 1-hydroxylase are parts of a common signaling pathway, or whether they can be influenced separately. An interesting recent novel finding in this context was that Janus kinase 3 (JAK3) may be involved in FGF23 signaling in renal proximal tubules, because global JAK3 knockout mice are characterized by increased circulating Fgf23 and vitamin D hormone, as well as increased urinary excretion of phosphate (Umbach et al., 2015). The latter observation underscores that the current knowledge about FGF23-induced intracellular signaling cascades in proximal tubular epithelial cells is still limited. A better understanding of the signaling mechanisms involved may lead to novel insights into the regulation of renal vitamin D hormone production and to new possibilities in the treatment of phosphate-wasting disorders.

There is also evidence that Klotho may regulate renal phosphate reabsorption by a FGF23 independent mechanism (Fig. 2). In this context, it was reported that Klotho can act as an autocrine phos-phaturic factor by altering the function of NaPi2a in renal proximal tubular epithelial cells by its putative glucuronidase activity (Hu et al., 2010). A problem in this model is that it requires the presence of functional Klotho protein in the urine, and it is currently unclear how Klotho crosses from the circulation or basolateral cell side to the urinary space. It was reported that soluble Klotho protein can be detected in murine urine (Chang et al., 2005; Hu et al., 2010). However, this is a controversial issue because our laboratory failed to find Klotho protein in mouse urine (Andrukhova et al., 2014c).

In distal renal tubules, soluble Klotho has been reported to act as a regulator of the epithelial calcium channel transient receptor

potential vanilloid-5 (TRPV5) (Chang et al., 2005). TRPV5 is a glycoprotein essential for apical entry of calcium in calcium-transporting renal epithelial cells, and apical membrane expression of fully glycosylated TRPV5 is the rate-limiting step in distal renal tubular transcellular calcium transport (Lambers et al., 2006). In this model (Fig. 2), Klotho, through its putative sialidase activity, promotes apical membrane abundance of TRPV5 by stabilizing the interaction between glycosylated TRPV5 and membrane-bound galectin, and by increasing the trafficking of TRPV5 towards the apical membrane (Chang et al., 2005; Cha et al., 2008; Wolf et al., 2014).

We recently discovered that FGF23 not only suppresses renal phosphate reabsorption in renal proximal tubules, but also regulates renal calcium and sodium handling in renal distal tubules (Andrukhova et al., 2014a, 2014c). We found that FGF23 signaling, acting through the basolateral FGFR/Klotho receptor complex, leads to an ERK1/2 and SGK1 dependent phosphorylation of with-no-lysine kinase 4 (WNK4) in renal distal tubular epithelium (Andrukhova et al., 2014a, 2014c). WNK 4 is a central molecule for trafficking of ion channels in renal epithelium (Jiang et al., 2007, 2008; Cha and Huang, 2010; McCormick et al., 2008; Bazua-Valenti and Gamba, 2015). WNK kinases regulate the intracellular transport of membrane proteins by acting as a complex of WNK1, 3, and 4 (McCormick et al., 2008).

FGF23-induced activation of WNK4 leads to increased apical membrane abundance of TRPV5 and of the Na+:Cl- cotransporter NCC in renal distal tubules which in turn results in increased cellular uptake of calcium and sodium (Andrukhova et al., 2014a, 2014c). Consequently, lack of Fgf23 and Klotho in Klotho and Fgf23 deficient mice causes renal calcium and sodium wasting, whereas injection of recombinant FGF23 decreases renal excretion of calcium and sodium (Andrukhova et al., 2014a, 2014c). Because sodium homeostasis is tightly coupled to the regulation of plasma volume and blood pressure, injection of normal mice with recombinant FGF23 resulted in plasma expansion, hypertension, and heart hypertrophy after only 5 days of treatment (Andrukhova et al., 2014a). The FGF23-induced hypertension was completely prevented by co-treatment with the NCC inhibitor chlorothiazide (Andrukhova et al., 2014a), showing that increased NCC-mediated renal sodium reabsorption plays a pivotal role in the FGF23-driven increase in blood pressure. Collectively, these data demonstrate that FGF23 is not only a phosphaturic, but also a calcium and sodium-conserving hormone (Fig. 1). This notion was recently corroborated by the demonstration of renal calcium wasting in conditional knockout mice in which distal tubular Fgf23 signaling was blocked by a specific deletion of Fgfrl in distal renal tubules (Han et al., 2016).

These findings may have major physiological and pathophysio-logical implications. It is well known that chronic hyper-phosphatemia is a risk factor for vascular calcification and cardiovascular disease (Dhingra et al., 2007; Scialla et al., 2013). FGF23 protects against the untoward biological consequences of hyperphosphatemia by increasing urinary phosphate excretion and downregulating vitamin D hormone production which indirectly reduces intestinal phosphate absorption. In a hyperphosphatemic situation, the calcium-conserving function of FGF23 may help to conserve calcium despite the suppression of vitamin D hormone synthesis by FGF23. It is interesting to note in this context that the other phosphaturic hormone, PTH, also combines a phosphaturic with a calcium-conserving function in the kidney.

In patients with chronic kidney disease (CKD), FGF23 and PTH are chronically elevated due to phosphate retention and decreased renal 1,25(OH)2D3 production. In this situation, the FGF23- and PTH-driven renal calcium conservation may contribute to the development of vascular calcification. In addition, it is well known

that circulating FGF23 is positively and dose-dependently associated with CKD progression, left ventricular hypertrophy, heart failure, vascular calcifications, and mortality in CKD patients (Isakova et al., 2011 ; Faul et al., 2011 ; Scialla et al., 2014). The novel link between FGF23 and volume regulation may provide a tentative explanation for this association. Another interesting observation was that FGF23 and aldosterone interact in the activation of SGK1 and in the regulation of NCC and ENaC in the distal nephron in mice (Andrukhova et al., 2014a). Surprisingly, we found that a low sodium diet and subsequently higher aldosterone levels aggravated the hypertensive effects of FGF23 (Andrukhova et al., 2014a). The likely explanation for this finding is that both FGF23 and aldoste-rone signaling converge on SGK1 in distal renal tubules which may lead to synergistic effects on NCC activation. Similar to FGF23, aldosterone activates SGK1, leading to increased membrane expression of ENaC (Chen et al., 1999) and activation of NCC through the SGK1 - WNK4 - STE20/SPS-1-related proline/alanine-rich kinase (SPAK) signaling axis (Rozansky et al., 2009; van der Lubbe et al., 2012; Ko et al., 2013). Aldosterone levels are typically elevated in CKD patients (Lattanzio and Weir, 2010). Hence, increased circulating aldosterone may additionally augment the effects of FGF23 on sodium retention in CKD patients.

Taken together, the hyperphosphatemia-driven increase in circulating FGF23 in CKD patients may further contribute to vascular calcifications, and may put additional strain on the heart by sodium and volume retention (Fig. 1). In agreement with this notion, aortic valve calcification was recently found to be positively associated with serum FGF23 and serum PTH in patients with CKD (Di Lullo et al., 2015).

3. Parathyroid gland

The parathyroid gland is a site of abundant Klotho expression (Kuro-o et al., 1997; Olauson et al., 2013). Several lines of evidence from in vivo and in vitro studies suggest that FGF23 suppresses PTH secretion (Ben Dov et al., 2007; Olauson et al., 2013). Because PTH stimulates FGF23 secretion in bone (Meir et al., 2014), the suppressive effects of FGF23 on PTH secretion form a negative feedback loop between bone and parathyroid gland (Fig. 1). However, the mechanism by which FGF23 regulates PTH secretion is not entirely clear. It was reported that Klotho interacts with Na+/K+-ATPase in parathyroid chief cells to stimulate PTH secretion under a hypo-calcemic challenge (Imura et al., 2007). However, this notion has later been challenged (Martuseviciene et al., 2011 ). Evidence from global and conditional Klotho knockout mice suggests that Klotho appears dispensable for the regulation of PTH secretion under steady state conditions: PTH serum levels were not different between global Klotho-1-/VDRd/d mice and VDRd/d control mice (Anour et al., 2012), and more recently Olauson and coworkers (Olauson et al., 2013) showed in a mouse model with a parathyroid-specific ablation of Klotho that Klotho deficiency was not associated with functional changes in the parathyroid glands, albeit some changes in the gene expression profile of chief cells were detected in the conditional knockout mice. Based on a combination of in vivo and in vitro experiments, the latter authors suggested that FGF23 suppresses PTH secretion by a Klotho independent pathway involving calcineurin (Olauson et al., 2013). On the other hand, several earlier studies suggested that a downregulation of Klotho and of FGFR induces resistance of the parathyroid gland to the FGF23-mediated suppression of PTH secretion in human CKD patients and experimental CKD models (Galitzer et al., 2010; Canalejo et al., 2010; Krajisnik et al., 2010; Komaba et al., 2010). In the heart, the Klotho independent FGF23 signaling is mediated through FGFR4 (Grabner et al., 2015). In the parathyroid, the FGFRs involved in Klotho dependent and independent suppressive actions of FGF23

on PTH secretion are still unclear. Therefore, the molecular mechanism of how FGF23 suppresses PTH secretion still awaits final clarification.

In addition, the relevance of the PTH-induced stimulation of FGF23 secretion and the FGF23-induced suppression of PTH secretion, which are both well documented in animal and in vitro models, for human physiology is presently unclear. For example, the increased FGF23 serum levels observed in patients with primary hyperparathyroidism may be an adaptive, indirect response to the PTH-induced increase in 1,25(OH)2D3 production (Witteveen et al., 2012). Furthermore, as mentioned above both PTH and FGF23 are typically elevated in patients with CKD, a finding which may be explained by parathyroid resistance to FGF23, but may alternatively call into question a robust suppression of PTH by FGF23 in humans.

4. Cardiovascular system

Although isolated reports about Klotho expression in human arteries and vascular smooth muscle cells exist (Lim et al., 2012), there is now general agreement that Klotho is neither expressed in the heart (Faul et al., 2011 ; Grabner et al., 2015) nor in blood vessels at physiologically significant levels (Scialla et al., 2013; Lindberg et al., 2013; Mencke et al., 2015). Nevertheless, as mentioned above, FGF23 is positively and dose-dependently associated with CKD progression, left ventricular hypertrophy, heart failure, vascular calcifications, and mortality in CKD patients (Juppner et al., 2010; Isakova et al., 2011; Faul et al., 2011; Scialla et al., 2014). In addition, FGF23 has been shown to be an independent risk factor for all-cause and cardiovascular mortality in patients with normal renal function undergoing coronary angiography (Brandenburg et al., 2014). The question is why?

There are several different explanatory scenarios. As mentioned above, increased circulating FGF23 may indirectly promote cardiovascular disease and renal disease progression by contributing to sodium and volume retention (Andrukhova et al., 2014a). Alternatively, there is good evidence that FGF23 can act directly on the heart. The recent reports by Faul et al. (2011) and Grabner et al. (2015) suggested that FGF23 induces left ventricular hypertrophy by a direct, Klotho independent, FGFR4-mediated action on car-diomyocytes. Moreover, local expression of FGF23 is increased in the heart of patients with CKD-induced left ventricular hypertrophy (Leifheit-Nestler et al., 2015), but also in rats and mice after induction of experimental myocardial infarction (Andrukhova et al., 2015). Therefore, FGF23 may also have a paracrine role in the pathogenesis of left ventricular hypertrophy.

It is still controversial whether FGF23 can directly act on blood vessels. Several groups were unable to find an effect of FGF23 on vascular calcification in vascular smooth muscle cells or organ cultures of different species (Lindberg et al., 2013; Scialla et al., 2013). In contrast, Jimbo et al. (2014) reported that FGF23 induced phosphate-induced vascular calcification in uremic rat aortic rings and rat vascular smooth muscle cells by a Klotho- and ERK1/2-dependent pathway. Since FGF23 suppresses vitamin D hormone production and since the vitamin D hormone is an important regulator of endothelial function (Andrukhova et al., 2014b), the effects of FGF23 on blood vessels in vivo may be indirect. However, FGF23 may also induce endothelial dysfunction by directly interfering with nitric oxide-mediated vasodilation (Silswal et al., 2014). Clearly, more work needs to be done to better define the putative role of FGF23 in blood vessels.

In agreement with the notion that the kidney is the main source of circulating soluble Klotho, the concentrations of soluble Klotho decline with declining kidney function and reduced renal functional mass (Hu et al., 2011 ; Pavik et al., 2013 ; Kitagawa et al., 2013). However, it is important to mention in this context that the

available assays for soluble Klotho are notoriously problematic. Nevertheless, it has been suggested that the CKD-associated deficiency in soluble Klotho is functionally linked to the development of heart hypertrophy (Xie et al., 2012) and vascular calcification (Hu et al., 2011). Furthermore, it has been shown that soluble Klotho is cardioprotective by an FGF23 independent downregulation of stress-induced calcium channels (Xie et al., 2012), and that soluble Klotho directly inhibits phosphate uptake in vascular smooth muscle cells (Hu et al., 2011) (Fig. 2). However, the link between soluble Klotho and cardiovascular risk was not substantiated by recent cohort studies: In contrast to circulating FGF23, soluble Klotho was neither found to be associated with cardiovascular risk in CKD patients (Seiler et al., 2014) nor in patients with normal renal function undergoing coronary angiography (Brandenburg et al., 2015).

5. Bone

Klotho is expressed at only very low levels in bone (Kuro-o et al., 1997; Miyagawa et al., 2014), whereas Fgf23 mRNA expression is distinctly higher in bone compared with other tissues (Yoshiko et al., 2007). Therefore, it is currently believed that osteoblasts and osteocytes are the major sources for circulating FGF23 in vivo (Martin et al., 2012). However, recent evidence has partially challenged this view because conditional ablation of Fgf23 in osteo-blasts and osteocytes using a Col1a1-Cre deleter mouse strain resulted in only about a 50% reduction in circulating intact Fgf23 levels (Clinkenbeard et al., 2016). This finding suggests that other cellular sources may contribute to circulating FGF23 levels in a significant fashion. In this context, it is important to note that Fgf23 mRNA expression is also found in other tissues than bone (Yoshiko et al., 2007).

It has long been known that bone mineralization is impaired in Klotho and Fgf23 deficient mice (Kuro-o et al., 1997; Shimada et al., 2004b; Sitara et al., 2004). Although the molecular mechanisms were only partially understood, earlier studies have shown that genetic ablation of vitamin D signaling largely rescues the mineralization defect seen in Fgf23~/~ and Klotho~/~ mice (Hesse et al., 2007; Anour et al., 2012). Moreover, ablation of osteopontin and PTH has been shown to partially rescue the osteomalacia in Fgß3~/ ~ and Klotho~/~ mice, respectively (Yuan et al., 2012, 2014).

We recently elucidated the mechanisms why bone mineralization is impaired in Fgf23~/~ and Klotho~/~ mice: the osteomalacia in Klotho deficient mice is solely caused by 1,25(OH)2D3-driven upregulation of the mineralization inhibitors pyrophosphate and osteopontin in bone (Murali et al., 2016). In agreement with the very low Klotho expression in bone, this finding suggests that Klotho lacks a 1,25(OH)2D3 independent role in bone mineralization. In contrast, we found that the mineralization defect in Fggf23~/ ~ mice is not only caused by a 1,25(OH)2D3-driven component similar to Klotho~/~ mice, but that lack of Fgf23 by itself contributed to the osteomalacia through regulating tissue nonspecific alkaline phosphatase (Tnap) and subsequently osteopontin expression (Murali et al., 2016). Our findings suggest that Fgf23 may act as an autocrine/paracrine mineralization-regulating factor in osteocytes and osteoblasts by suppressing Tnap transcription via a Klotho independent FGFR3-mediated signaling pathway (Murali et al., 2016). Therefore, Fgf23 deficiency leads to an upregulation of Tnap expression and increased phosphate production which in turn stimulates osteopontin secretion (Murali et al., 2016). Conversely, treatment of osteoblasts with recombinant FGF23 suppresses TNAP, and subsequently leads to accumulation of the TNAP substrate pyrophosphate (Murali et al., 2016). Although the latter mechanism needs further in vivo confirmation in gain-of-function models, it may have major implications for the mineralization defects seen in

diseases characterized by excessive osteocytic FGF23 secretion such as X-linked hypophosphatemic rickets or CKD, because increased FGF23 concentrations in bone may inhibit mineralization through suppression of TNAP and subsequent accumulation of pyrophosphate (Fig. 1).

6. Central nervous system

The choroid plexus is a site of abundant Klotho expression (Kuro-o et al., 1997), and FGF23 was actually first described in the mouse brain (Yamashita et al., 2000). However, very little is known about the functional role of Klotho and FGF23 in the central nervous system. Klotho/VDR and Fg/23/VDR compound mutant mice do not have obvious behavioral abnormalities (Anour et al., 2012; Streicher et al., 2012). Therefore, it is unlikely that both proteins have a major function in the brain. However, it has never been tested in detail whether absence of Klotho or Fgf23 may cause more subtle neurological phenotypes. It is interesting to note in this context that the secreted form of Klotho was recently found to be inversely associated with aging and Alzheimer's disease in mice (Masso et al., 2015). Moreover, mice overexpressing Klotho show enhanced cognition (Dubal et al., 2014), and are partially protected against cognitive decline and premature mortality in transgenic models of Alzheimer's disease (Dubal et al., 2015). In addition, certain natural variations in the Klotho gene are associated with bigger brain volume and enhanced cognition in human populations (Dubal et al., 2014; Yokoyama et al., 2015). However, these observations in humans and mice currently lack a mechanistic explanation.

7. Conclusion

Recent advances in the field of FGF23 and Klotho biology have revealed major new functions of FGF23 and Klotho signaling in the kidney, in the heart, in bone, in blood vessels, and in the parathyroid gland. It is now clear that FGF23 is far more than only a phosphaturic bone-derived hormone. Rather, FGF23 has emerged as a pleiotropic endocrine and auto-/paracrine factor not only involved in phosphate homeostasis, but also in calcium and sodium metabolism, in bone mineralization as well as in the development of cardiac hypertrophy. These novel findings have linked phosphate with volume homeostasis, and may have major pathophysiological implications for chronic kidney disease, cardiovascular diseases, and disorders of bone mineralization.

Financial support and sponsorship

This work was supported by a grant from the Austrian Science Fund (FWF 24186-B21) to R.G.E.

Conflicts of interest

The author declares no conflicts of interest. Acknowledgements

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