Klotho in chronic kidney disease--What's new?

Klotho, named after a Greek goddess who spins the thread of life, was identified in 1997 as a gene mutated in a mouse strain that developed a premature ageing syndrome [ 1 ]. A defect in Klotho gene expression in mice results in shortened life span, growth retardation, hypogonadism, accelerated thymic involution, skin atrophy, muscle atrophy, vascular calcification, osteopaenia, pulmonary emphysema, cognition impairment [ 2 ], hearing loss [ 3 ] and motor neuron degeneration [ 4 ] among others. These ageing-like phenotypes are associated with elevated serum levels of 1,25-dihydroxyvitamin D 3 , phosphate and calcium [ 5 ]. The Klotho gene encodes a single-pass transmembrane protein that belongs to a family 1 glycosidase [ 6 ] and is expressed primarily in renal tubules. Function of the Klotho protein was not clear at that time. Fibroblast growth factor-23 (FGF23) was cloned based on sequence similarity to the other members of FGF ligand superfamily [ 7 ] and identified in 2000 as a gene mutated in patients with autosomal dominant hypophosphataemic rickets (ADHR) [ 8 ]. Unlike classical FGF ligands that function as paracrine and/or autocrine factors, FGF23 functions as an endocrine factor [ 9 ]. FGF23 is primarily produced in osteocytes and acts on the kidney to induce phosphate excretion (phosphaturic hormone) and suppress serum levels of 1,25-dihydroxyvitamin D 3 (counter-regulatory hormone for vitamin D) [ 10 ]. Patients with ADHR exhibit increased serum FGF23 levels due to mutations in the FGF23 gene that confer resistance to proteolytic degeneration of FGF23 protein [ 11 ]. However, the identity of the FGF23 receptor was not clear because the affinity of FGF23 to any known FGF receptors is extremely low in vitro [ 12 ]. Klotho and FGF23, seemingly unrelated proteins, had been studied independently until it was realized that Klotho-deficient mice [ 1 ] and FGF23-deficient mice [ 13 ] shared identical phenotypes. FGF23-deficient mice also develop multiple ageing-like phenotypes associated with hyperphosphataemia, hypercalcaemia and hypervitaminosis D. We reported in 2006 that Klotho formed a complex with several FGF receptor isoforms (FGFR1c, 3c, 4) and significantly increased the affinity of FGF23 to the FGF receptors [ 14 ]. Thus, Klotho functions as an obligatory co-receptor for FGF23. This finding was later confirmed in an independent study [ 15 ]. The fact that FGF23 requires Klotho as a co-receptor explains why Klotho-deficient mice develop phenotypes identical with those observed in FGF23-deficient mice and why Klotho-deficient mice had extremely high serum FGF23 levels [ 15 ]. In addition, kidney-specific expression of Klotho explains why FGF23 can identify the kidney as its target organ among many other tissues that express multiple FGFR isoforms. The bone–kidney endocrine axis mediated by FGF23 and Klotho has emerged as an essential component in the regulation of phosphate homeostasis. Serum phosphate levels are determined by a counterbalance between absorption of dietary phosphate from the intestine, mobilization from bone (the reservoir of phosphate and calcium) and excretion from the kidney into urine [ 16 ]. When phosphate is in excess, FGF23 is secreted from bone and acts on the kidney where Klotho is expressed. As a phosphaturic hormone, FGF23 reduces the amount of sodium phosphate co-transporter type-2a (NaPi-2a) on the brush border membrane of proximal tubules, thereby promoting renal phosphate excretion. As a counter-regulatory hormone for vitamin D, FGF23 suppresses synthesis and promotes inactivation of 1,25-dihydroxyvitamin D 3 in proximal tubules. FGF23 suppresses expression of the Cyp27b1 gene that encodes 1α-hydroxylase, the enzyme that converts vitamin D from an inactive form (25-hydroxyvitamin D 3 ) to the active form (1,25-dihydroxyvitamin D 3 ). In addition, FGF23 increases expression of the Cyp24 gene that encodes 24-hydroxylase, the enzyme that inactivates 1,25-dihydroxyvitamin D 3 . The ability of FGF23 to reduce serum 1,25-dihydroxyvitamin D 3 levels also contributes to induce a negative phosphate balance through limiting phosphate absorption from the intestine. Importantly, 1,25-dihydroxyvitamin D 3 induces expression of the FGF23 gene and closes a negative feedback loop (Figure 1 ). It should be noted that Klotho expression is much more abundant in distal convoluted tubules than in proximal tubules, whereas both phosphate reabsorption and vitamin D synthesis take place in proximal tubules. It remains to be determined whether FGF23 acts on proximal tubules directly or acts on distal convoluted tubules to generate a signal to proximal tubules that suppresses phosphate reabsorption and vitamin D synthesis. A recent study supports the latter possibility. Despite the fact that the proximal tubule primarily expresses FGFR3 but not the other FGFR isoforms, knockout of the Fgfr3 gene in Hyp mice, which have elevated serum FGF23 levels, failed to correct the phosphate-wasting syndrome in Hyp mice [ 17 ]. These findings suggest that activity of FGF23 is independent of FGF signalling activation in the proximal tubule. The bone–kidney–parathyroid endocrine axes mediated by FGF23 and Klotho. Active vitamin D (1,25-dihydroxyvitamin D 3 ) binds to the vitamin D receptor (VDR) in osteocytes. The ligand-bound VDR forms a heterodimer with a nuclear receptor RXR and transactivates the FGF23 gene expression. FGF23 secreted from bone acts on the Klotho–FGFR complex in kidney (the bone–kidney axis) and parathyroid gland (the bone–parathyroid axis). In kidney, FGF23 suppresses synthesis of active vitamin D by down-regulating expression of the Cyp27b1 gene and promotes its inactivation by up-regulating expression of the Cyp24 gene, thereby closing a negative feedback loop for vitamin D homeostasis. In the parathyroid gland, FGF23 suppresses production and secretion of PTH. Since PTH is a potent inducer of Cyp27b1 gene expression, suppression of PTH by FGF23 reduces expression of the Cyp27b1 gene as well as serum levels of 1,25-dihydroxyvitamin D 3 , which closes another long negative feedback loop for vitamin D homeostasis. Klotho and FGF23 are indispensable for the regulation of vitamin D metabolism because defects in either Klotho or FGF23 cause hypervitaminosis D. Parathyroid hormone (PTH) plays an important role not only in calcium metabolism but also in phosphate homeostasis. In contrast to FGF23, PTH induces expression of the Cyp27b1 gene and increases serum 1,25-dihydroxyvitamin D 3 levels. Recent studies showed that the parathyroid gland expresses Klotho endogenously, suggesting that parathyroid may be a target organ of FGF23. In fact, FGF23 suppresses expression and secretion of PTH both in vivo and in vitro [ 18,19 ]. Thus, the ability of FGF23 to reduce serum PTH levels may further enhance the activity of FGF23 as a counter-regulatory hormone for vitamin D and contribute to a long negative feedback loop involving bone, kidney and parathyroid gland (Figure 1 ). Disruption of the bone–kidney endocrine axis mediated by Klotho and FGF23 results in hyperphosphataemia, hypercalcaemia and hypervitaminosis D associated with multiple ageing-like phenotypes. These observations have raised the possibility that toxicity of phosphate, calcium and/or vitamin D may be responsible for the premature ageing syndrome observed in Klotho- and FGF23-deficient mice. Several studies have addressed this possibility. First, a vitamin D-deficient diet not only restored serum phosphate and calcium levels but also rescued many ageing-like phenotypes including vascular calcification in Klotho-deficient mice [ 20 ]. Second, ablation of vitamin D activity in FGF23-deficient mice by disrupting the Cyp27b1 gene [ 21 ] or vitamin D receptor gene [ 22 ] also rescued both hyperphosphataemia and the premature ageing syndrome. Lastly, low-phosphate diet rescued shortened life span and vascular calcification in FGF23-deficient mice [ 23 ]. These studies have provided unequivocal evidence that the premature ageing syndrome caused by defects in the bone–kidney endocrine axis is due to the toxicity of phosphate, calcium and/or vitamin D. Of note, low-phosphate diet rescued FGF23-deficient mice despite the fact that it further increased already elevated serum calcium and vitamin D levels [ 23 ], suggesting that phosphate, but not calcium or vitamin D, is toxic when retained and may be primarily responsible for the ageing-like phenotypes. It remains to be determined whether high serum vitamin D and/or calcium levels are a prerequisite for phosphate to induce ageing-like phenotypes. Recent epidemiological studies support the notion of ‘phosphate toxicity’ in humans. Serum phosphate levels were shown to positively correlate all-cause mortality risk, even when serum phosphate levels are within the normal range [ 24 ]. In addition, chronic kidney disease (CKD) patients with hyperphosphataemia (≥6.5 mg/dl) were reported to have higher risk for death resulting from several diseases including coronary artery disease than those with the lower serum phosphate levels (<6.5 mg/dl) [ 25 ]. Based on these observations, controlling serum phosphate levels below 6.5 mg/dl has become an important therapeutic goal for CKD. Thus, low-phosphate diet and/or phosphate binders have been increasingly recognized as important therapeutic options for preventing life-threatening complications of CKD. The National Kidney Foundation task force indicated that the cardiovascular mortality of a 35-year-old patient on dialysis was equivalent to that of an 80-year-old healthy individual, rendering CKD to be the most potent accelerator of vascular senescence [ 26 ]. Furthermore, the American Heart Association announced in 2003 that CKD should be included in the highest risk group for cardiovascular disease [ 27 ]. Like Klotho-deficient mice, CKD patients suffer vascular calcification and have elevated serum levels of FGF23 and phosphate. Importantly, Klotho expression is decreased in CKD patients [ 28 ]. These observations suggest that Klotho deficiency may contribute to pathophysiology of CKD. Of note, recent animal studies have shown that Klotho functions as a renoprotective factor. Although the mechanism remains to be determined, over-expression of Klotho ameliorated progressive renal injury in mouse models of glomerulonephritis [ 29 ] and acute kidney injury [ 30 ]. Thus, decrease in Klotho expression potentially accelerates renal damage, leading to a deterioration spiral of Klotho expression and renal function. Because 1,25-dihydroxyvitamin D 3 increases Klotho expression in kidney [ 20 ], vitamin D treatment may be useful for interrupting this vicious cycle. Epidemiological studies have identified high serum levels of phosphate and FGF23 as independent mortality risks in CKD patients [ 31 ]. Importantly, serum FGF23 levels increase before serum phosphate levels increase during the progression of CKD [ 32 ], suggesting that resistance to FGF23 may be one of the earliest changes in phosphate metabolism in CKD. Although the mechanism of FGF23 resistance is yet to be determined, it can be caused by a decrease in renal Klotho expression. Provided that serum FGF23 levels are a surrogate marker for renal Klotho expression levels, the fact that high serum FGF23 levels are associated with poor prognosis in patients undergoing dialysis [ 31 ] suggests that low renal Klotho expression levels may be primarily responsible for the poor prognosis. It remains to be determined whether renal Klotho expression levels reflect functional nephron mass that can respond to FGF23. It has become increasingly clear that phosphate metabolism plays a critical role in the pathophysiology in CKD and that hyperphosphataemia should be aggressively treated to improve life expectancy of CKD patients. In this context, the bone–kidney–parathyroid endocrine axis mediated by Klotho and FGF23 is expected to be a novel target of therapeutic intervention. Conflict of interest statement . None declared.