A major breakthrough in systemic phosphate homeostasis regulation was achieved by the demonstration of strikingly similar physical, morphological, and biochemical phenotypes of fibroblast growth factor 23 (Fgf23) and klotho ablated mice, which led to identification of klotho as an Fgf23 signaling cofactor. Here, we generated Fgf23 and klotho double-knockout (Fgf23(-/-)/klotho(-/-)) mice to test the hypothesis whether Fgf23 has a klotho-independent function. Fgf23(-/-)/klotho(-/-) mice are viable and have high serum phosphate levels, similar to Fgf23(-/-) and klotho(-/-) single-knockout mice. In addition, the Fgf23(-/-)/klotho(-/-) mice have increased renal expression of the sodium/phosphate cotransporter NaP(i)2a and of 1- alpha-hydroxylase concomitant with increased serum levels of 1,25-dihydroxyvitamin-D, as also observed in the Fgf23(-/-) and klotho(-/-) mice. Moreover, Fgf23(-/-)/klotho(-/-) mice show soft tissue and vascular calcification, severe muscle wasting, hypogonadism, pulmonary emphysema, distention of intestinal wall, and skin atrophy, all of which are also seen in Fgf23(-/-) and klotho(-/-) mice. Notably, injection of bioactive FGF23 protein into Fgf23(-/-)/klotho(-/-) and klotho(-/-) mice does not lower serum phosphate, whereas in wild-type and Fgf23(-/-) mice, it reduces serum phosphate. Together, these results provide compelling evidence that Fgf23 does not have a klotho-independent role in the regulation of systemic phosphate and vitamin D homeostasis.

Identifying factors that accelerate the aging process can provide important therapeutic targets for slowing down this process. Misregulation of phosphate homeostasis has been noted in various skeletal, cardiac, and renal diseases, but the exact role of phosphate toxicity in mammalian aging is not clearly defined. Phosphate is widely distributed in the body and is involved in cell signaling, energy metabolism, nucleic acid synthesis, and the maintenance of acid-base balance by urinary buffering. In this study, we used an in vivo genetic approach to determine the role of phosphate toxicity in mammalian aging. Klotho-knockout mice (klotho(-/-)) have a short life span and show numerous physical, biochemical, and morphological features consistent with premature aging, including kyphosis, uncoordinated movement, hypogonadism, infertility, severe skeletal muscle wasting, emphysema, and osteopenia, as well as generalized atrophy of the skin, intestine, thymus, and spleen. Molecular and biochemical analyses suggest that increased renal activity of sodium-phosphate cotransporters (NaPi2a) leads to severe hyperphosphatemia in klotho(-/-) mice. Genetically reducing serum phosphate levels in klotho(-/-) mice by generating a NaPi2a and klotho double-knockout (NaPi2a(-/-)/klotho(-/-)) strain resulted in amelioration of premature aging-like features. The NaPi2a(-/-)/klotho(-/-) double-knockout mice regained reproductive ability, recovered their body weight, reduced their organ atrophy, and suppressed ectopic calcifications, with the resulting effect being prolonged survival. More important, when hyperphosphatemia was induced in NaPi2a(-/-)/klotho(-/-) mice by feeding with a high-phosphate diet, premature aging-like features reappeared, clearly suggesting that phosphate toxicity is the main cause of premature aging in klotho(-/-) mice. The results of our dietary and genetic manipulation studies provide in vivo evidence for phosphate toxicity accelerating the aging process and suggest a novel role for phosphate in mammalian aging.

Changes in the expression of klotho, a β-glucuronidase, contribute to the development of features that resemble those of premature aging, as well as chronic renal failure. Klotho knockout mice have increased expression of the sodium/phosphate cotransporter (NaPi2a) and 1α-hydroxylase in their kidneys, along with increased serum levels of phosphate and 1,25-dihydroxyvitamin D. These changes are associated with widespread soft-tissue calcifications, generalized tissue atrophy, and a shorter lifespan in the knockout mice. To determine the role of the increased vitamin D activities in klotho knockout animals, we generated klotho and 1α-hydroxylase double-knockout mice. These double mutants regained body weight and developed hypophosphatemia with a complete elimination of the soft-tissue and vascular calcifications that were routinely found in klotho knockout mice. The markedly increased serum fibroblast growth factor 23 and the abnormally low serum parathyroid hormone levels, typical of klotho knockout mice, were significantly reversed in the double-knockout animals. These in vivo studies suggest that vitamin D has a pathologic role in regulating abnormal mineral ion metabolism and soft-tissue anomalies of klotho-deficient mice.

Hyp mice possess a mutation that inactivates the phosphate-regulating gene, which is homologous to the endopeptidases of the X-chromosome (PHEX). The mutation is associated with severe hypophosphatemia due to excessive urinary phosphate wasting. Such urinary phosphate wasting in Hyp mice is associated with an increased serum accumulation of fibroblast growth factor (FGF) 23. We wanted to determine the biological significance of increased serum FGF23 levels and concomitant hypophosphatemia in Hyp mice and to evaluate whether FGF23 activity could be modified by manipulating klotho (a cofactor of FGF23 signaling). We generated Hyp and klotho double-mutant mice (Hyp/klotho(-/-)). Severe hypophosphatemia of Hyp mice was reversed to hyperphosphatemia in Hyp/klotho(-/-) double mutants, despite the fact that the double mutants showed significantly increased serum levels of FGF23. Hyperphosphatemia in Hyp/klotho(-/-) mice was associated with increased renal expression of sodium/phosphate cotransporter 2a (NaPi2a) protein. Exogenous injection of bioactive parathyroid hormone 1-34 down-regulated renal expression of NaPi2a and consequently reduced serum levels of phosphate in Hyp/klotho(-/-) mice. Moreover, in contrast to the Hyp mice, the Hyp/klotho(-/-) mice showed significantly higher serum levels of 1,25-dihydroxyvitamin D and developed extensive calcification in soft tissues and vascular walls. Furthermore, compared with the Hyp mice, Hyp/klotho(-/-) mice were smaller in size, showed features of generalized tissue atrophy, and generally died by 15-20 wk of age. Our in vivo studies provide genetic evidence for a pathological role of increased FGF23 activities in regulating abnormal phosphate homeostasis in Hyp mice. Moreover, these results suggest that even when serum levels of FGF23 are significantly high, in the absence of klotho, FGF23 is unable to regulate systemic phosphate homeostasis. Our in vivo observations have significant clinical implications in diseases associated with increased FGF23 activity and suggest that the functions of FGF23 can be therapeutically modulated by manipulating the effects of klotho.

Summary:From April 1998 to March 2000, a cytomegalovirus (CMV) antigenemia-guided pre-emptive approach for CMV disease was evaluated in 77 adult patients who received allogeneic hematopoietic stem cell transplantation at the National Cancer Center Hospital. A CMV antigenemia assay was performed at least once a week after engraftment. High-level antigenemia was defined as a positive result with 10 or more positive cells per 50 000 cells and low-level antigenemia was defined as less than 10 positive cells. Among the 74 patients with initial engraftment, 51 developed positive antigenemia. Transplantation from alternative donors and the development of grade II-IV GVHD were independent risk factors for positive antigenemia. Ganciclovir was administered as pre-emptive therapy in 39 patients in a risk-adapted manner. None of the nine low-risk patients with low-level antigenemia as their initial positive result developed high-level antigenemia even though ganciclovir was withheld. Only one patient developed early CMV disease (hepatitis) during the study period. CMV antigenemia resolved in all but two cases, in whom ganciclovir was replaced with foscarnet. In eight patients, however, the neutrophil count decreased to 0.5 × 10 9 /l or less after starting ganciclovir, including three with documented infections and two with subsequent secondary graft failure. The total amount of ganciclovir and possibly the duration of high-dose ganciclovir might affect the incidence of neutropenia. We concluded that antigenemia-guided pre-emptive therapy with a decreased dose of ganciclovir and response-oriented dose adjustment might be appropriate to decrease the toxicity of ganciclovir without increasing the risk of CMV disease. Bone Marrow Transplantation (2001) 27, 437-444.

Summary:To evaluate the efficacy of long-term administration of acyclovir as prophylaxis against varicella-zoster virus (VZV) reactivation, we analyzed the medical records of 86 consecutive adult patients who obtained engraftment after allogeneic hematopoietic stem cell transplantation from January 1996 to March 2000. We started longterm low-dose (400 mg/day) oral administration of acyclovir in June 1999, and this was continued until the end of immunosuppressive therapy after transplantation. There was no breakthrough reactivation of VZV in patients receiving acyclovir. Five patients who were receiving cyclosporine or prednisolone developed VZV reactivation after discontinuing acyclovir. With this prophylaxis, the cumulative incidence of VZV reactivation at 1 year after transplantation decreased from 33% to 10% (P = 0.025). On multivariate analysis, the use of long-term acyclovir was identified as a significant independent parameter for the development of VZV reactivation. These findings suggest the efficacy of longterm prophylaxis with low-dose acyclovir. Resumption of acyclovir upon restarting immunosuppressive therapy might be important for the further prevention of VZV reactivation. The benefit of long-term low-dose acyclovir should be confirmed prospectively. Bone Marrow Transplantation (2001) 28, 689-692.

e It has been recently established that Klotho coreceptors associate with fibroblast growth factor (FGF) receptor tyrosine kinases (FGFRs) to enable signaling by endocrine-acting FGFs. However, the molecular interactions leading to FGF-FGFR-Klotho ternary complex formation remain incompletely understood. Here, we show that in contrast to ␣Klotho, ␤Klotho binds its cognate endocrine FGF ligand (FGF19 or FGF21) and FGFR independently through two distinct binding sites. FGF19 and FGF21 use their respective C-terminal tails to bind to a common binding site on ␤Klotho. Importantly, we also show that Klotho coreceptors engage a conserved hydrophobic groove in the immunoglobulin-like domain III (D3) of the "c" splice isoform of FGFR. Intriguingly, this hydrophobic groove is also used by ligands of the paracrine-acting FGF8 subfamily for receptor binding. Based on this binding site overlap, we conclude that while Klotho coreceptors enhance binding affinity of FGFR for endocrine FGFs, they actively suppress binding of FGF8 subfamily ligands to FGFR. Fibroblast growth factor (FGF) signaling plays pleiotropic roles in metazoan development and metabolism (5, 24). Based on sequence homology and phylogenetic and structural considerations, the 18 mammalian FGF ligands are grouped into five paracrine-acting subfamilies and one endocrine-acting subfamily comprising FGF19, FGF21, and FGF23 (24,43). Paracrine FGFs have high affinity for pericellular heparan sulfate (HS) glycosaminoglycans (3, 5) and form distinct morphogenetic gradients in the pericellular matrix (27, 39) to fulfill essential roles during embryonic development (7,24,65). In contrast, endocrine FGFs exhibit poor affinity for HS (3, 15) and thus are able to enter the blood circulation to regulate key metabolic processes, including bile acid homeostasis (18,20,36) and hepatic glucose and protein metabolism (30, 57) (FGF19), glucose and lipid metabolism (4, 21, 29, 58) (FGF21), and vitamin D and phosphate homeostasis (1, 59, 62) (FGF23). These hormone-like FGFs have taken center stage in drug discovery for a number of inherited and acquired metabolic disorders (5, 6).Mammalian FGFs signal through four FGF receptor (FGFR) tyrosine kinases (FGFR1 to FGFR4) and their alternatively spliced isoforms (23, 43). The extracellular domain of a prototypical FGFR consists of three immunoglobulin-like domains (D1 to D3), and structural and biochemical studies have established that the region including D2, D3, and the D2-D3 linker comprises the minimal ligand-binding domain (54,55,63). The ligand-binding specificity of FGFR1 to FGFR3 is primarily regulated by a tissuespecific splicing in D3 of these receptors that generates "b" and "c" isoforms (9,26,42,52). Structural studies have revealed that this splicing alters the primary amino acid sequence of key ligandbinding loops/pockets in D3 (50,74).In addition to mediating paracrine FGF gradient formation in the pericellular matrix, HS is required for enhancing the affinity of paracrine FGF for FGFR and promoting dimerization of ligan...

Background-Klotho-knockout mice (klotho Ϫ/Ϫ ) have increased renal expression of sodium/phosphate cotransporters (NaPi2a), associated with severe hyperphosphatemia. Such serum biochemical changes in klotho Ϫ/Ϫ mice lead to extensive soft-tissue anomalies and vascular calcification. To determine the significance of increased renal expression of the NaPi2a protein and concomitant hyperphosphatemia and vascular calcification in klotho Ϫ/Ϫ mice, we generated klotho and NaPi2a double-knockout (klotho Ϫ/Ϫ /NaPi2a Ϫ/Ϫ ) mice. Methods and Results-Genetic inactivation of NaPi2a activity from klotho Ϫ/Ϫ mice reversed the severe hyperphosphatemia to mild hypophosphatemia or normophosphatemia. Importantly, despite significantly higher serum calcium and 1,25-dihydroxyvitamin D levels in klotho /NaPi2aϪ/Ϫ mice, the vascular and soft-tissue calcifications were reduced. Extensive soft-tissue anomalies and cardiovascular calcification were consistently noted in klotho Ϫ/Ϫ mice by 6 weeks of age; however, these vascular and soft-tissue abnormalities were absent even in 12-week-old double-knockout mice. Klotho Ϫ/Ϫ /NaPi2a Ϫ/Ϫ mice also regained body weight and did not develop the generalized tissue atrophy often noted in klotho Ϫ/Ϫ single-knockout mice. Conclusion-Our in vivo genetic manipulation studies have provided compelling evidence for a pathological role of increased NaPi2a activities in regulating abnormal mineral ion metabolism and soft-tissue anomalies in klotho Ϫ/Ϫ mice. Notably, our results suggest that serum phosphate levels are the important in vivo determinant of calcification and that lowering serum phosphate levels can reduce or eliminate soft-tissue and vascular calcification, even in presence of extremely high serum calcium and 1,25-dihydroxyvitamin D levels. These in vivo observations have significant clinical importance and therapeutic implications for patients with chronic kidney disease with cardiovascular calcification. (Circ Cardiovasc Genet. 2009;2:583-590.) Key Words: klotho Ⅲ vitamin D Ⅲ NaPi2a Ⅲ calcification U nderstanding the molecular regulation of phosphate homeostasis has enormous clinical and biological importance because it is involved in numerous essential biochemical reactions, including cell signaling process and energy metabolism. Adequate bone mineralization is closely dependent on the status of phosphate metabolism. Abnormal regulation of phosphate homeostasis can cause myopathy, cardiac dysfunctions, hematologic abnormalities, and vascular and soft-tissue calcifications. 1-3 Recent studies have found that klotho, a transmembrane protein, is actively involved in regulation of mineral ion metabolism by affecting the functionality of ion channels and cotransporter proteins in the kidney. 4,5 The in vivo importance of klotho in regulation of mineral ion metabolism is further evident in klotho-knockout mice because these mice have severely impaired mineral ion homeostasis. 5,6 Clinical Perspective on p 590The klotho Ϫ/Ϫ mice develop severe hyperphosphatemia by 3 weeks of age and remain hyperph...