A new gene, termed klotho, has been identified that is involved in the suppression of several ageing phenotypes. A defect in klotho gene expression in the mouse results in a syndrome that resembles human ageing, including a short lifespan, infertility, arteriosclerosis, skin atrophy, osteoporosis and emphysema. The gene encodes a membrane protein that shares sequence similarity with the beta-glucosidase enzymes. The klotho gene product may function as part of a signalling pathway that regulates ageing in vivo and morbidity in age-related diseases.

Chronic kidney disease (CKD) is a public health epidemic that increases risk of death due to cardiovascular disease. Left ventricular hypertrophy (LVH) is an important mechanism of cardiovascular disease in individuals with CKD. Elevated levels of FGF23 have been linked to greater risks of LVH and mortality in patients with CKD, but whether these risks represent causal effects of FGF23 is unknown. Here, we report that elevated FGF23 levels are independently associated with LVH in a large, racially diverse CKD cohort. FGF23 caused pathological hypertrophy of isolated rat cardiomyocytes via FGF receptor-dependent activation of the calcineurin-NFAT signaling pathway, but this effect was independent of klotho, the coreceptor for FGF23 in the kidney and parathyroid glands. Intramyocardial or intravenous injection of FGF23 in wild-type mice resulted in LVH, and klotho-deficient mice demonstrated elevated FGF23 levels and LVH. In an established animal model of CKD, treatment with an FGF-receptor blocker attenuated LVH, although no change in blood pressure was observed. These results unveil a klotho-independent, causal role for FGF23 in the pathogenesis of LVH and suggest that chronically elevated FGF23 levels contribute directly to high rates of LVH and mortality in individuals with CKD.

A defect in Klotho gene expression in mice accelerates the degeneration of multiple age-sensitive traits. Here, we show that overexpression of Klotho in mice extends life span. Klotho protein functions as a circulating hormone that binds to a cell-surface receptor and represses intracellular signals of insulin and insulin-like growth factor 1 (IGF1), an evolutionarily conserved mechanism for extending life span. Alleviation of aging-like phenotypes in Klotho-deficient mice was observed by perturbing insulin and IGF1 signaling, suggesting that Klotho-mediated inhibition of insulin and IGF1 signaling contributes to its anti-aging properties. Klotho protein may function as an anti-aging hormone in mammals.Klotho was originally identified as a mutated gene in a mouse strain that accelerates agedependent loss of function in multiple age-sensitive traits (1). An insertional mutation that disrupts the 5′ promoter region of the Klotho gene resulted in a strong hypomorphic allele. Mice homozygous for the mutated allele (KL −/− mice) appeared normal until 3 to 4 weeks old but then began to manifest multiple age-related disorders observed in humans, including ectopic calcification, skin atrophy, muscle atrophy, osteoporosis, arteriosclerosis, and pulmonary emphysema. KL −/− mice suffered premature death around two months of age.

The Klotho gene encodes a 130-kDa single-pass transmembrane protein with a short cytoplasmic domain (10 amino acids) and is expressed predominantly in the kidney. Mice carrying a loss-of-function mutation in the Klotho gene develop a syndrome resembling human aging, including shortened life span, skin atrophy, muscle atrophy, osteoporosis, arteriosclerosis, and pulmonary emphysema (1). Conversely, overexpression of the Klotho gene extends the life span and increases resistance to oxidative stress in mice (2-4). These observations suggest that the Klotho gene functions as an aging suppressor gene. The extracellular domain of Klotho protein is shed and secreted in the blood (2, 5), potentially functioning as a humoral factor that signals suppression of intracellular insulin/IGF1 signaling, which partly contributes to its anti-aging properties (2). However, a signaling pathway(s) directly activated by Klotho protein, including the identity of the Klotho receptor, has not been determined. The function of the transmembrane form of Klotho protein also remains to be determined.Fibroblast growth factor-23 (FGF23) 2 was originally identified as a gene mutated in patients with autosomal dominant hypophosphatemic rickets (6), where mutations in the FGF23 gene conferred resistance to inactivation by protease cleavage, resulting in elevated serum levels of FGF23 (7-12). FGF23 inhibits phosphate transport in renal proximal tubular cells and in proximal tubules perfused in vitro (13). Consistent with these findings, mice defective in FGF23 expression show increased renal phosphate reabsorption and hyperphosphatemia (14). Although FGF23 binds to multiple FGF receptors (FGFRs) (15), it has modest receptor affinity (K D ϭ 200 -700 nM) and often requires cofactors such as heparin or glycosaminoglycan (15, 16) to activate FGF signaling in cultured cells and to inhibit phosphate transport in proximal tubules perfused in vitro (13).Klotho-deficient mice (Klotho Ϫ/Ϫ mice) and FGF23 deficient mice (Fgf23 Ϫ/Ϫ mice) develop many common phenotypes, including shortened life span, growth retardation, infertility, muscle atrophy, hypoglycemia, and vascular calcification in the kidneys. Notably, they both have increased serum levels of phosphate (14, 17). These observations have led us to the hypothesis that Klotho and FGF23 may function via a common signal transduction pathway. In this report we show that Klotho binds to multiple FGFRs and functions as a cofactor necessary for FGF signaling activation by FGF23. MATERIALS AND METHODSExpression Vectors-Complementary DNA containing the mouse FGFRs coding region (IMAGE Clone, Invitrogen, supplemental Fig. 1) were cloned into pcDNA3.1(ϩ) expression vector (Invitrogen). Before subcloning, a V5-epitope tag was added to the C terminus and appropriate restriction enzyme sites to the both ends using synthetic oligonucleotides and polymerase chain reaction. Expression vectors for the mouse FGF23 resistant to proteolytic inactivation (R179Q) (18), the transmembrane form of mouse Klotho, and the extracel...

Soft-tissue calcification is a prominent feature in both chronic kidney disease (CKD) and experimental Klotho deficiency, but whether Klotho deficiency is responsible for the calcification in CKD is unknown. Here, wild-type mice with CKD had very low renal, plasma, and urinary levels of Klotho. In humans, we observed a graded reduction in urinary Klotho starting at an early stage of CKD and progressing with loss of renal function. Despite induction of CKD, transgenic mice that overexpressed Klotho had preserved levels of Klotho, enhanced phosphaturia, better renal function, and much less calcification compared with wild-type mice with CKD. Conversely, Klotho-haploinsufficient mice with CKD had undetectable levels of Klotho, worse renal function, and severe calcification. The beneficial effect of Klotho on vascular calcification was a result of more than its effect on renal function and phosphatemia, suggesting a direct effect of Klotho on the vasculature. In vitro, Klotho suppressed Na ϩ -dependent uptake of phosphate and mineralization induced by high phosphate and preserved differentiation in vascular smooth muscle cells. In summary, Klotho is an early biomarker for CKD, and Klotho deficiency contributes to soft-tissue calcification in CKD. Klotho ameliorates vascular calcification by enhancing phosphaturia, preserving glomerular filtration, and directly inhibiting phosphate uptake by vascular smooth muscle. Replacement of Klotho may have therapeutic potential for CKD.

The fibroblast growth factor (FGF) 19 subfamily of ligands, FGF19, FGF21, and FGF23, function as hormones that regulate bile acid, fatty acid, glucose, and phosphate metabolism in target organs through activating FGF receptors (FGFR1-4). We demonstrated that Klotho and ␤Klotho, homologous single-pass transmembrane proteins that bind to FGFRs, are required for metabolic activity of FGF23 and FGF21, respectively. Here we show that, like FGF21, FGF19 also requires ␤Klotho. Both FGF19 and FGF21 can signal through FGFR1-3 bound by ␤Klotho and increase glucose uptake in adipocytes expressing FGFR1. Additionally, both FGF19 and FGF21 bind to the ␤Klotho-FGFR4 complex; however, only FGF19 signals efficiently through FGFR4. Accordingly, FGF19, but not FGF21, activates FGF signaling in hepatocytes that primarily express FGFR4 and reduces transcription of CYP7A1 that encodes the rate-limiting enzyme for bile acid synthesis. We conclude that the expression of ␤Klotho, in combination with particular FGFR isoforms, determines the tissue-specific metabolic activities of FGF19 and FGF21.

Phosphate homeostasis is maintained by a counterbalance between efflux from the kidney and influx from intestine and bone. FGF23 is a bone-derived phosphaturic hormone that acts on the kidney to increase phosphate excretion and suppress biosynthesis of vitamin D. FGF23 signals with highest efficacy through several FGF receptors (FGFRs) bound by the transmembrane protein Klotho as a coreceptor. Since most tissues express FGFR, expression of Klotho determines FGF23 target organs. Here we identify the parathyroid as a target organ for FGF23 in rats. We show that the parathyroid gland expressed Klotho and 2 FGFRs. The administration of recombinant FGF23 led to an increase in parathyroid Klotho levels. In addition, FGF23 activated the MAPK pathway in the parathyroid through ERK1/2 phosphorylation and increased early growth response 1 mRNA levels. Using both rats and in vitro rat parathyroid cultures, we show that FGF23 suppressed both parathyroid hormone (PTH) secretion and PTH gene expression. The FGF23-induced decrease in PTH secretion was prevented by a MAPK inhibitor. These data indicate that FGF23 acts directly on the parathyroid through the MAPK pathway to decrease serum PTH. This bone-parathyroid endocrine axis adds a new dimension to the understanding of mineral homeostasis.

Although members of the fibroblast growth factor (FGF) family and their receptors have well-established roles in embryogenesis, their contributions to adult physiology remain relatively unexplored. Here, we use real-time quantitative PCR to determine the mRNA expression patterns of all 22 FGFs, the seven principal FGF receptors (FGFRs), and the three members of the Klotho family of coreceptors in 39 different mouse tissues. Unsupervised hierarchical cluster analysis of the mRNA expression data reveals that most FGFs and FGFRs fall into two groups the expression of which is enriched in either the central nervous system or reproductive and gastrointestinal tissues. Interestingly, the FGFs that can act as endocrine hormones, including FGF15/19, FGF21, and FGF23, cluster in a third group that does not include any FGFRs, underscoring their roles in signaling between tissues. We further show that the most recently identified Klotho family member, Lactase-like, is highly and selectively expressed in brown adipose tissue and eye and can function as an additional coreceptor for FGF19. This FGF atlas provides an important resource for guiding future studies to elucidate the physiological functions of FGFs in adult animals.