Fibroblast growth factor 23–Klotho : un nouvel axe de régulation du bilan du phosphate

The kidney is a key player of phosphate balance, it determines serum phosphate levels by coupling phosphate reabsorption in the renal proximal tubule, calcitriol synthesis and consequently intestinal phosphate absorption. The identification of fibroblast growth factor 23 (FGF23) as a hormone regulating phosphate and calcitriol metabolism has unveiled the mechanisms that coordinate these renal proximal tubule functions. A bone–kidney axis has emerged that controls bone mineralization. Animal model studies have improved our understanding of phosphate homeostasis and revealed the role of the protein Klotho, which is mandatory to FGF23 action. In this review we detail FGF23 and Klotho implications in physiology and in genetic or acquired disorders. Phosphate ion is involved in vascular and soft tissue calcification and is important for cell proliferation. Disorders of FGF23–Klotho axis alter life-span and the survival in some cancers.     

The bone and the kidney

Renal tubular diseases may present with osteopenia, osteoporosis or osteomalacia, as a result of significant derangements in body electrolytes. In case of insufficient synthesis of calcitriol, as in renal failure, the more complex picture of renal osteodystrophy may develop. Hypothetically, also disturbed renal production of BMP-7 and Klotho could cause bone disease. However, the acknowledgment that osteocytes are capable of producing FGF23, a phosphaturic hormone at the same time modulating renal synthesis of calcitriol, indicates that it is also bone that can influence renal function. Importantly, a feed-back mechanism exists between FGF23 and calcitriol synthesis, while Klotho, produced by the kidney, determines activity and selectivity of FGF23. Identification of human diseases linked to disturbed production of FGF23 and Klotho underlines the importance of this new bone-kidney axis. Kidney and bone communicate reciprocally to regulate the sophisticated machinery responsible for divalent ions homeostasis and for osseous or extraosseous mineralisation processes.     

FGF23, alpha-Klotho and vitamin D mediated calcium-phosphate metabolism in haemodialysis patients

BackgroundKlotho is a prote˝in that acts as a co-receptor for FGF23. FGF23-Klotho axis has great importance regarding the regulation of mineral metabolism by kidneys. In this study, we analysed FGF23, Klotho, 1,25-dihydroxyvitamin D3, 25-hydroxyvitamin D, parathormone, Calcium and Phosphate levels of haemodialysis patients in order to investigate the nature of the mineral metabolism disruption in chronic kidney diseases.MethodsSixty haemodialysis patients and 34 healthy controls were included in the study. Serum iFGF, cFGF, and soluble Klotho were analysed using ELISA kits. Moreover, 1,25-dihydroxyvitamin D3 was determined using LCMS/MS. Calcium, phosphate, iPTH and 25-hydroxyvitamin D were measured using autoanalyzers.ResultsIn haemodialysis patients, iFGF23, cFGF23, iPTH and P levels were significantly higher, and 1,25-dihydroxyvitamin D3, Klotho and Ca levels were significantly lower compared with the control group. There was no significant difference in the 25-hydroxyvitamin D levels.ConclusionsOur study showed that lack of sufficient amounts of Klotho is crucial for mineral metabolism disruptions seen as a complication of chronic kidney diseases. Despite the high levels of the hormone, FGF23 is unable to accomplish its function properly, likely due to deteriorated kidney function in haemodialysis patients.     

Significance of the anti-aging protein Klotho

Abstract

The Klotho gene was identified as an ‘aging suppressor' in mice. Overexpression of the Klotho gene extends lifespan and defective Klotho results in rapid aging and early death. Both the membrane and secreted forms of Klotho have biological activity that include regulatory effects on general metabolism and a more specific effect on mineral metabolism that correlates with its effect on aging. Klotho serves as a co-receptor for fibroblast growth factor (FGF), but it also functions as a humoral factor that regulates cell survival and proliferation, vitamin D metabolism, and calcium and phosphate homeostasis and may serve as a potential tumor suppressor. Moreover, Klotho protects against several pathogenic processes in a FGF23-independent manner. These processes include cancer metastasis, vascular calcification, and renal fibrosis. This review covers the recent advances in Klotho research and discusses novel Klotho-dependent mechanisms that are clinically relevant in aging and age-related diseases.     

Conversion of a paracrine fibroblast growth factor into an endocrine fibroblast growth factor

FGFs 19, 21, and 23 are hormones that regulate in a Klotho co-receptor-dependent fashion major metabolic processes such as glucose and lipid metabolism (FGF21) and phosphate and vitamin D homeostasis (FGF23). The role of heparan sulfate glycosaminoglycan in the formation of the cell surface signaling complex of endocrine FGFs has remained unclear. Here we show that heparan sulfate is not a component of the signal transduction unit of FGF19 and FGF23. In support of our model, we convert a paracrine FGF into an endocrine ligand by diminishing heparan sulfate-binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor-binding site to home the ligand into the target tissue. In addition to serving as a proof of concept, the ligand conversion provides a novel strategy for engineering endocrine FGF-like molecules for the treatment of metabolic disorders, including global epidemics such as type 2 diabetes and obesity.     

Renal Expression of FGF23 in Progressive Renal Disease of Diabetes and the Effect of Ace Inhibitor

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone mainly produced by bone that acts in the kidney through FGF receptors and Klotho. Here we investigated whether the kidney was an additional source of FGF23 during renal disease using a model of type 2 diabetic nephropathy. Renal expression of FGF23 and Klotho was assessed in Zucker diabetic fatty (ZDF) and control lean rats at 2, 4, 6, 8 months of age. To evaluate whether the renoprotective effect of angiotensin converting enzyme (ACE) inhibitor in this model was associated with changes in FGF23 and Klotho, ZDF rats received ramipril from 4, when proteinuric, to 8 months of age. FGF23 mRNA was not detectable in the kidney of lean rats, nor of ZDF rats at 2 months of age. FGF23 became measurable in the kidney of diabetic rats at 4 months and significantly increased thereafter. FGF23 protein localized in proximal and distal tubules. Renal Klotho mRNA and protein decreased during time in ZDF rats. As renal disease progressed, serum phosphate levels increased in parallel with decline of fractional phosphorus excretion. Ramipril limited proteinuria and renal injury, attenuated renal FGF23 upregulation and ameliorated Klotho expression. Ramipril normalized serum phosphate levels and tended to increase fractional phosphorus excretion. These data indicate that during progressive renal disease the kidney is a site of FGF23 production which is limited by ACE inhibition. Interfering pharmacologically with the delicate balance of FGF23 and phosphorus in diabetes may have implications in clinics.     

The cooperation of FGF receptor and Klotho is involved in excretory canal development and regulation of metabolic homeostasis in Caenorhabditis elegans

FGFs have traditionally been associated with cell proliferation, morphogenesis, and development; yet, a subfamily of FGFs (FGF19, -21, and -23) functions as hormones to regulate glucose, lipid, phosphate, and vitamin D metabolism with impact on energy balance and aging. In mammals, Klotho and beta-Klotho are type 1 transmembrane proteins that function as obligatory co-factors for endocrine FGFs to bind to their cognate FGF receptors (FGFRs). Mutations in Klotho/beta-Klotho or fgf19, -21, or -23 are associated with a number of human diseases, including autosomal dominant hypophosphatemic rickets, premature aging disorders, and diabetes. The Caenorhabditis elegans genome contains two paralogues of Klotho/beta-Klotho, klo-1, and klo-2. klo-1 is expressed in the C. elegans excretory canal, which is structurally and functionally paralogous to the vertebrate kidney. KLO-1 associates with EGL-15/FGFR, suggesting a role for KLO-1 in the fluid homeostasis phenotype described previously for egl-15/fgfr mutants. Altered levels of EGL-15/FGFR signaling lead to defects in excretory canal development and function in C. elegans. These results suggest an evolutionarily conserved function for the FGFR-Klotho complex in the development of excretory organs such as the mammalian kidney and the worm excretory canal. These results also suggest an evolutionarily conserved function for the FGFR-Klotho axis in metabolic regulation.     

FGF23 promotes renal calcium reabsorption through the TRPV5 channel

αKlotho is thought to activate the epithelial calcium channel Transient Receptor Potential Vanilloid‐5 (TRPV5) in distal renal tubules through its putative glucuronidase/sialidase activity, thereby preventing renal calcium loss. However, αKlotho also functions as the obligatory co‐receptor for fibroblast growth factor‐23 (FGF23), a bone‐derived phosphaturic hormone. Here, we show that renal calcium reabsorption and renal membrane abundance of TRPV5 are reduced in Fgf23 knockout mice, similar to what is seen in αKlotho knockout mice. We further demonstrate that αKlotho neither co‐localizes with TRPV5 nor is regulated by FGF23. Rather, apical membrane abundance of TRPV5 in renal distal tubules and thus renal calcium reabsorption are regulated by FGF23, which binds the FGF receptor‐αKlotho complex and activates a signaling cascade involving ERK1/2, SGK1, and WNK4. Our data thereby identify FGF23, not αKlotho, as a calcium‐conserving hormone in the kidney.     

Antibody-Mediated Activation of FGFR1 Induces FGF23 Production and Hypophosphatemia

The phosphaturic hormone Fibroblast Growth Factor 23 (FGF23) controls phosphate homeostasis by regulating renal expression of sodium-dependent phosphate co-transporters and cytochrome P450 enzymes involved in vitamin D catabolism. Multiple FGF Receptors (FGFRs) can act as receptors for FGF23 when bound by the co-receptor Klotho expressed in the renal tubular epithelium. FGFRs also regulate skeletal FGF23 secretion; ectopic FGFR activation is implicated in genetic conditions associated with FGF23 overproduction and hypophosphatemia. The identity of FGFRs that mediate the activity of FGF23 or that regulate skeletal FGF23 secretion remains ill defined. Here we report that pharmacological activation of FGFR1 with monoclonal anti-FGFR1 antibodies (R1MAb) in adult mice is sufficient to cause an elevation in serum FGF23 and mild hypophosphatemia. In cultured rat calvariae osteoblasts, R1MAb induces FGF23 mRNA expression and FGF23 protein secretion into the culture medium. In a cultured kidney epithelial cell line, R1MAb acts as a functional FGF23 mimetic and activates the FGF23 program. siRNA-mediated Fgfr1 knockdown induced the opposite effects. Taken together, our work reveals the central role of FGFR1 in the regulation of FGF23 production and signal transduction, and has implications in the pathogenesis of FGF23-related hypophosphatemic disorders.     

Direct,Acute Effects of Klotho and FGF23 on Vascular Smooth Muscle and Endothelium

Chronic kidney disease (CKD) is regarded as a state of Klotho deficiency and FGF23 excess. In patients with CKD a strong association has been found between increased serum FGF23 and mortality risk, possibly via enhanced atherosclerosis, vascular stiffness, and vascular calcification. The aim of this study was to examine the hypothesis that soluble Klotho and FGF23 exert direct, rapid effects on the vessel wall. We used three in vitro models: mouse aorta rings, human umbilical vein endothelial cells, and human vascular smooth muscle cells (HVSMC). Increasing medium concentrations of soluble Klotho and FGF23 both stimulated aorta contractions and increased ROS production in HVSMC. Klotho partially reverted FGF23 induced vasoconstriction, induced relaxation on phosphate preconstricted aorta and enhanced endothelial NO production in HUVEC. Thus Klotho increased both ROS production in HVSMC and NO production in endothelium. FGF23 induced contraction in phosphate preconstricted vessels and increased ROS production. Phosphate, Klotho and FGF23 together induced no change in vascular tone despite increased ROS production. Moreover, the three compounds combined inhibited relaxation despite increased NO production, probably owing to the concomitant increase in ROS production. In conclusion, although phosphate, soluble Klotho and FGF23 separately stimulate aorta contraction, Klotho mitigates the effects of phosphate and FGF23 on contractility via increased NO production, thereby protecting the vessel to some extent against potentially noxious effects of high phosphate or FGF23 concentrations. This novel observation is in line with the theory that Klotho deficiency is deleterious whereas Klotho sufficiency is protective against the negative effects of phosphate and FGF23 which are additive.     

C-terminal tail of FGF19 determines its specificity toward Klotho co-receptors

FGF19 subfamily proteins (FGF19, FGF21, and FGF23) are unique members of fibroblast growth factors (FGFs) that regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis in an endocrine fashion. Their activities require the presence of alpha or betaKlotho, two related single-pass transmembrane proteins, as co-receptors in relevant target tissues. We previously showed that FGF19 can bind to both alpha and betaKlotho, whereas FGF21 and FGF23 can bind only to either betaKlotho or alphaKlotho, respectively in vitro. To determine the mechanism regulating the binding and specificity among FGF19 subfamily members to Klotho family proteins, chimeric proteins between FGF19 subfamily members or chimeric proteins between Klotho family members were constructed to probe the interaction between those two families. Our results showed that a chimera of FGF19 with the FGF21 C-terminal tail interacts only with betaKlotho and a chimera with the FGF23 C-terminal tail interacts only with alphaKlotho. FGF signaling assays also reflected the change of specificity we observed for the chimeras. These results identified the C-terminal tail of FGF19 as a region necessary for its recognition of Klotho family proteins. In addition, chimeras between alpha and betaKlotho were also generated to probe the regions in Klotho proteins that are important for signaling by this FGF subfamily. Both FGF23 and FGF21 require intact alpha or betaKlotho for signaling, respectively, whereas FGF19 can signal through a Klotho chimera consisting of the N terminus of alphaKlotho and the C terminus of betaKlotho. Our results provide the first glimpse of the regions that regulate the binding specificity between this unique family of FGFs and their co-receptors.     

Familial tumoral calcinosis and the role of O-glycosylation in the maintenance of phosphate homeostasis

Familial tumoral calcinosis refers to a group of disorders inherited in an autosomal recessive fashion. Hyperphosphatemic tumoral calcinosis is characterized by increased re-absorption of phosphate through the renal proximal tubule, resulting in elevated phosphate concentration and deposition of calcified deposits in cutaneous and subcutaneous tissues, as well as, occasionally, in visceral organs. The disease was found to result from mutations in at least 3 genes: GALNT3, encoding a glycosyltransferase termed ppGalNacT3, FGF23 encoding a potent phosphaturic protein, and KL encoding Klotho. Recent data showed that ppGalNacT3 mediates O-glycosylation of FGF23, thereby allowing for its secretion and possibly protecting it from proteolysis-mediated inactivation. Klotho was found to serve as a co-receptor for FGF23, thereby integrating the genetic data into a single physiological system. The elucidation of the molecular basis of HFTC shed new light upon the mechanisms regulating phosphate homeostasis, suggesting innovative therapeutic strategies for the management of hyperphosphatemia in common acquired conditions such as chronic renal failure.     

FGF23 and disorders of phosphate homeostasis

It is well known that fibroblast growth factor (FGF) family members are associated with embryonic development and are critical for basic metabolic functions. This review will focus upon fibroblast growth factor-23 (FGF23) and its roles in disorders associated with phosphate handling. The discovery that mutations in FGF23 were responsible for the isolated renal phosphate wasting disorder autosomal dominant hypophosphatemic rickets (ADHR) has ascribed novel functions to the FGF family. FGF23 circulates in the bloodstream, and animal models demonstrate that FGF23 controls phosphate and Vitamin D homeostasis through the regulation of specific renal proteins. The ADHR mutations in FGF23 produce a protein species less susceptible to proteolytic processing. X-linked hypophosphatemic rickets (XLH), tumor-induced osteomalacia (TIO), and fibrous dysplasia of bone (FD) are disorders involving phosphate homeostasis that share phenotypes with ADHR, indicating that FGF23 may be a common denominator for the pathophysiology of these syndromes. Our understanding of FGF23 will help to develop novel therapies for phosphate wasting disorders, as well as for disorders of increased serum phosphate, such as tumoral calcinosis, a rare disorder, and renal failure, a common disorder.     

Deletion of PTH rescues skeletal abnormalities and high osteopontin levels in Klotho-/- mice

Maintenance of normal mineral ion homeostasis is crucial for many biological activities, including proper mineralization of the skeleton. Parathyroid hormone (PTH), Klotho, and FGF23 have been shown to act as key regulators of serum calcium and phosphate homeostasis through a complex feedback mechanism. The phenotypes of Fgf23(-/-) and Klotho(-/-) (Kl(-/-)) mice are very similar and include hypercalcemia, hyperphosphatemia, hypervitaminosis D, suppressed PTH levels, and severe osteomalacia/osteoidosis. We recently reported that complete ablation of PTH from Fgf23(-/-) mice ameliorated the phenotype in Fgf23(-/-)/PTH(-/-) mice by suppressing serum vitamin D and calcium levels. The severe osteomalacia in Fgf23(-/-) mice, however, persisted, suggesting that a different mechanism is responsible for this mineralization defect. In the current study, we demonstrate that deletion of PTH from Kl(-/-) (Kl(-/-)/PTH(-/-) or DKO) mice corrects the abnormal skeletal phenotype. Bone turnover markers are restored to wild-type levels; and, more importantly, the skeletal mineralization defect is completely rescued in Kl(-/-)/PTH(-/-) mice. Interestingly, the correction of the osteomalacia is accompanied by a reduction in the high levels of osteopontin (Opn) in bone and serum. Such a reduction in Opn levels could not be observed in Fgf23(-/-)/PTH(-/-) mice, and these mice showed sustained osteomalacia. This significant in vivo finding is corroborated by in vitro studies using calvarial osteoblast cultures that show normalized Opn expression and rescued mineralization in Kl(-/-)/PTH(-/-) mice. Moreover, continuous PTH infusion of Kl(-/-) mice significantly increased Opn levels and osteoid volume, and decreased trabecular bone volume. In summary, our results demonstrate for the first time that PTH directly impacts the mineralization disorders and skeletal deformities of Kl(-/-), but not of Fgf23(-/-) mice, possibly by regulating Opn expression. These are significant new perceptions into the role of PTH in skeletal and disease processes and suggest FGF23-independent interactions of PTH with Klotho.     

Arterial Klotho Expression and FGF23 Effects on Vascular Calcification and Function

Recent studies support a role for FGF23 and its co-receptor Klotho in cardiovascular pathology, yet the underlying mechanisms remain largely elusive. Herein, we analyzed the expression of Klotho in mouse arteries and generated a novel mouse model harboring a vascular smooth muscle cell specific deletion of Klotho (Sm22-KL^−/−). Arterial Klotho expression was detected at very low levels with quantitative real-time PCR; Klotho protein levels were undetectable by immunohistochemistry and Western blot. There was no difference in arterial Klotho between Sm22-KL^−/− and wild-type mice, as well as no changes in serum markers of mineral metabolism. Intravenous delivery of FGF23 elicited a rise in renal (0.005; p<0.01) but not arterial Egr-1 expression, a marker of Klotho-dependent FGF23 signaling. Further, the impact of FGF23 on vascular calcification and endothelial response was evaluated in bovine vascular smooth muscle cells (bVSMC) and in a murine ex vivo model of endothelial function, respectively. FGF23 treatment (0.125–2 ng/mL) did not modify calcification in bVSMCs or dilatory, contractile and structural properties in mice arterial specimen ex vivo. Collectively, these results demonstrate that FGF23-Klotho signaling is absent in mouse arteries and that the vascular response was unaffected by FGF23 treatment. Thus, our data do not support Klotho-mediated FGF23 effects in the vasculature although confirmative studies in humans are warranted.     

Regulation of fibroblast growth factor-23 signaling by klotho

The aging suppressor gene Klotho encodes a single-pass transmembrane protein. Klotho-deficient mice exhibit a variety of aging-like phenotypes, many of which are similar to those observed in fibroblast growth factor-23 (FGF23)-deficient mice. To test the possibility that Klotho and FGF23 may function in a common signal transduction pathway(s), we investigated whether Klotho is involved in FGF signaling. Here we show that Klotho protein directly binds to multiple FGF receptors (FGFRs). The Klotho-FGFR complex binds to FGF23 with higher affinity than FGFR or Klotho alone. In addition, Klotho significantly enhanced the ability of FGF23 to induce phosphorylation of FGF receptor substrate and ERK in various types of cells. Thus, Klotho functions as a cofactor essential for activation of FGF signaling by FGF23.     

Deregulated Renal Calcium and Phosphate Transport during Experimental Kidney Failure

Impaired mineral homeostasis and inflammation are hallmarks of chronic kidney disease (CKD), yet the underlying mechanisms of electrolyte regulation during CKD are still unclear. Here, we applied two different murine models, partial nephrectomy and adenine-enriched dietary intervention, to induce kidney failure and to investigate the subsequent impact on systemic and local renal factors involved in Ca²⁺ and P_i regulation. Our results demonstrated that both experimental models induce features of CKD, as reflected by uremia, and elevated renal neutrophil gelatinase-associated lipocalin (NGAL) expression. In our model kidney failure was associated with polyuria, hypercalcemia and elevated urinary Ca²⁺ excretion. In accordance, CKD augmented systemic PTH and affected the FGF23-αklotho-vitamin-D axis by elevating circulatory FGF23 levels and reducing renal αklotho expression. Interestingly, renal FGF23 expression was also induced by inflammatory stimuli directly. Renal expression of Cyp27b1, but not Cyp24a1, and blood levels of 1,25-dihydroxy vitamin D₃ were significantly elevated in both models. Furthermore, kidney failure was characterized by enhanced renal expression of the transient receptor potential cation channel subfamily V member 5 (TRPV5), calbindin-D_28k, and sodium-dependent P_i transporter type 2b (NaP_i2b), whereas the renal expression of sodium-dependent P_i transporter type 2a (NaP_i2a) and type 3 (PIT2) were reduced. Together, our data indicates two different models of experimental kidney failure comparably associate with disturbed FGF23-αklotho-vitamin-D signalling and a deregulated electrolyte homeostasis. Moreover, this study identifies local tubular, possibly inflammation- or PTH- and/or FGF23-associated, adaptive mechanisms, impacting on Ca²⁺/P_i homeostasis, hence enabling new opportunities to target electrolyte disturbances that emerge as a consequence of CKD development.