The Antisenescence Protein Klotho Is Necessary for FGF23-Induced Phosphaturia

It was almost one decade ago (1) that the klotho gene had been identified, but it does not fail to still spring surprises. Mice with a defect of klotho gene expression secondary to an insertional mutation disrupting the 5′ region develop a phenotype that reproduces not only individual features of human aging, but a complex spectrum of premature senescence: short lifespan, growth retardation, infertility, premature thymic involution, arteriosclerosis, skin atrophy, muscle atrophy, osteoporosis, pulmonary emphysema, ectopic calcification, motor neuron degeneration, cognition impairment, hearing disorder, and others (1). Conversely, overexpression of klotho extended the lifespan of mice (2). These observations suggest that the klotho gene acts as an aging suppressor gene. The link of the protein to the lifespan of mice led to the somewhat strange name “klotho.” Klotho was a Greek goddess, one of the three Parca, who was thought to spin the thread of life from her distaff onto her spindle. This mythology led Johannes Brahms to his famous romantic “Gesang der Parzen” (Schicksalslied; song of destiny). The klotho gene is not only a determinant of premature senescence in mice. In humans as well, features related to senescence such as lifespan, osteoporosis, stroke, coronary disease are correlated to polymorphisms of the klotho gene (3,4). The klotho gene codes for a 130-kDa single-pass transmembrane protein with a short 10-amino acid cytoplasmic domain and a large extracellular domain that is shed and secreted into the blood (2,5). Although the Klotho protein is expressed in several tissues, the quantitatively most important site is the distal convoluted tubule in the kidney (1,6), but it is not yet certain whether Klotho acts in the kidney in a paracrine or endocrine fashion (in this case it would be another renal hormone). How does klotho prolong lifespan? The klotho mouse with deficient klotho (klotho−/−) is hypoglycemic and extremely sensitive to insulin (7), whereas transgenic mice that overexpress klotho are insulin-resistant. Insulin resistance caused by genetic manipulation of insulin receptors or postreceptor steps is known to be associated with lifespan extension in a long list of species from Caenorhabditis elegans or Drosophila to mammalians such as the mouse (8). A second feature of the Klotho mouse is increased oxidative stress (9). But in the context of the kidney, the relation of Klotho to mineral and vitamin D metabolism is of greatest interest. The FGF23 gene was originally identified as the gene mutated in autosomal dominant hypophosphatemic rickets (10,11). FGF23 codes for a phosphaturic hormone (11) that also accounts for other forms of hypophosphatemia (12) and is synthesized mainly by osteoblastic cells (13). The striking observation has been made that klotho knockout (1) and FGF23 knockout mice (14,15) share a great number of features such as short lifespan, growth retardation, hypogonadism, premature thymic involution, skin atrophy, muscle atrophy, arteriosclerosis, osteoporosis, pulmonary emphysema, soft tissue calcification, and, of particular interest in the context of the above study, hyperphosphatemia and increased 1,25(OH)2 D3 (14–16). FGF23 inhibits not only phosphate reabsorption in the proximal tubule, but also regulates the activities of the key enzymes controlling vitamin D metabolism in the kidney, ie, CYP27B1 (1α-hydroxylase) and CYP24A1 (24-hydroxylase) (17). FGF23 deficiency in contrast increases renal phosphate reabsorption and increases 1,25(OH)2 D3 (14,15). Simultaneous knockout of FGF23 plus 1α-hydroxylase abrogated hyperphosphatemia and tissue calcification and rescued many premature aging-like features of the FGF23 knockout mice (18). This observation suggests an important role of active vitamin D in the genesis of the FGF23 knockout phenotype (6,19). Previous studies have shown that FGF23 not only inhibits phosphate reabsorption via the sodium phosphate transporter (NaPi2a) in the brush border membranes of the proximal tubule, but also shares common signalling pathways with Klotho (20). The Klotho protein binds to multiple FGF receptors. Furthermore, the Klotho-FGF receptor complex binds FGF23 with higher affinity than the FGF receptor or Klotho alone, which suggests that FGF23 requires Klotho to activate FGF signalling, thus explaining why klotho knockout mice develop many features of the phenotype observed in FGF23 mice. The above paper (21) now carries this issue one step further by clarifying in considerable detail the signalling pathways involved. In a nutshell they showed that the Klotho protein is required to convert a FGF receptor that interacts broadly with many FGF isoforms (a canonical receptor) into a receptor highly specific for FGF23. The authors discovered that injection of FGF23 into mice triggered early events in the kidney but not in other organs: phosphorylation of extracellular signal-regulated kinase (ERK) within the ultrashort period of 10 minutes; upregulation of the expression of early growth-responsive gene 1 (Egr1) was seen and used as a readout within 1 hour. The authors now pursued several strategies. First, in homogenates of mouse kidney they tried to identify the molecule(s) directly binding to FGF23. To this end the homogenate was run through FGF23-Sepharose; matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry revealed that the protein bound was Klotho. When klotho was transiently expressed in Chinese hamster ovary (CHO) or other cells, high and low affinity binding sites for FGF23 emerged and the cells responded by phosphorylating ERK and upregulating Egr-1. As was already known, klotho knockout mice had elevated serum phosphate, 1,25(OH)2 D3, and calcium, as do FGF knockout mice. The authors now showed that–presumably in response to hyperphosphatemia–FGF23 concentrations were massively increased (ie, several thousand-fold) in the klotho knockout mice. This observation resembles the increased FGF23 concentration that is seen in uremic patients in response to phosphate retention (12). To strengthen the evidence, the authors used a complementary approach, and to this end they first produced a monoclonal antibody against the extracellular portion of mouse Klotho protein. The monoclonal antibody blocked the FGF23-induced increase in Egr-1 promoter activity as a readout in cells expressing Klotho. To prove that the monoclonal antibody against Klotho abrogated the interaction between FGF23 and Klotho in vivo, the monoclonal antibody was injected into wild-type mice. The vitamin D–regulating enzymes in the kidney changed in the direction seen in FGF23 knockouts (14,15): the mRNA for CYP27B1 increased and the mRNA for CYP24A1 decreased; in parallel with an increased abundance of the sodium–phosphate cotransporter (NaPi2a) in the renal brush border membranes, serum phosphate increased—possibly the result of increased 1,25(OH)2 D3—serum calcium increased as well. Thus, blocking Klotho with the monoclonal antibody reproduced the main features of the FGF23 knockout mouse, which indicates that interaction between Klotho and FGF23 is necessary for FGF23 to express its activity in the kidney. The study thus identified another player in the genesis of the disturbed mineral metabolism of renal disease. The unfolding evidence indicates, in retrospect, that the simplifying assumption of the past, ie, that the disturbance of the mineral metabolism in renal failure was the result of abnormal phosphate and calcium concentration and balance (22), requires revision and sophistication in the light of findings obtained with the powerful methodologies available today.