Klotho

Ectopic calcification of the vasculature comes with age and is also prevalent in patients with chronic kidney disease (CKD). Klotho, a protein highly expressed in the kidney, has been identified as a novel anti-aging factor,1,2 and Klotho-deficient animals develop medial vascular calcification.3 Importantly, patients with CKD are deficient in Klotho, suggesting that lack of this protein may be a key factor driving calcification. To date, the mechanisms whereby Klotho protects against vascular calcification have focused on its role as an obligate co-factor for fibroblast growth factor 23 (FGF23) signaling in regulating phosphate and vitamin D metabolism. However, emerging evidence suggests that Klotho may also exert direct effects on vascular smooth muscle cells (VSMC) and act as a novel circulating inhibitor of vascular calcification. klotho is named after the Greek Goddess of Fate, who spins the thread of life. Mice deficient in Klotho are progeric and display tissue-specific, age-associated defects, including medial vascular calcification.3 Klotho is highly expressed in the kidney and an essential co-factor for the phosphatonin FGF23, which is expressed in bone. Klotho-FGF23 signaling promotes renal phosphate excretion through sodium phosphate (NaPi) co-transporter 2a channels, thereby lowering blood phosphate levels, and downregulates the expression of 1α-hydroxylase to suppress production of active 1,25-dihydroxyvitamin D. Defects in either FGF23 or Klotho cause a combination of metabolic disturbances, including hyperphosphatemia, hypercalcemia, and hypervitaminosis D.4–6 FGF23 levels are upregulated in early CKD, possibly as an adaptive response to promote phosphate excretion. However, with progressive CKD, Klotho expression significantly decreases and signaling is disabled.7,8 Importantly, the resultant hyperphosphatemia, a universal feature of advanced CKD, is a potent inducer of VSMC calcification in vitro and in vivo9,10 and is independently associated with increased cardiovascular mortality.11 To date, accumulated evidence has led to the contention that Klotho exerts all of its effects on vascular calcification indirectly through its actions on these key metabolic processes. Animal studies show that the life span and the aging-like phenotypes of Klotho or FGF23-deficient mice can be mostly rescued by a vitamin D–deficient diet or by resolving hyperphosphatemia with dietary or genetic manipulation. Thus, as in CKD, vascular calcification in these mice is thought to be due to VSMC damage resulting from chronically high phosphate and, to a lesser extent, dysregulated calcium (Ca) and vitamin D levels.4 However, an exciting new study published in this issue of JASN puts a different slant on these observations.12 Hu et al.12 set out to explore whether Klotho itself was a direct inhibitor of VSMC calcification. Using genetically manipulated mice expressing varying levels of Klotho and in vitro tools, they provide evidence that Klotho may also exert direct effects on VSMCs, and this may be an additional mechanism for protection against calcification. The first key observation made in this study was that murine CKD induced by 5/6 nephrectomy was associated with a marked reduction in plasma, urine, and kidney Klotho and that calcification progressed in these animals in the same way as in Klotho haploinsufficient animals. The decrease in urinary Klotho in patients with CKD7 led them to hypothesize that Klotho deficiency is responsible for the calcification observed in CKD. To test this further, they induced CKD in mice expressing various levels of Klotho. As expected, they found that Klotho deficiency exacerbated the effects of CKD on vascular calcification, whereas increased Klotho expression ameliorated these effects. Not surprising, Klotho deficiency was associated with elevated parathyroid hormone, increased 1,25-dihydroxyvitamin D, and elevated circulating inorganic phosphate (Pi), whereas overexpression of Klotho normalized these factors, supporting the conclusion from previous studies that vascular calcification is due to systemic perturbations of Pi and calciotropic hormones.4 However, when they divided the mice into subgroups on the basis of their genetic Klotho status and then correlated tissue Ca content with plasma creatinine (Cr) and Pi levels, they found for a given Pi or Cr level that animals with the highest Klotho levels had the lowest Ca content. This relationship was most dramatic in the kidney, where Klotho is known to have direct effects on organ function4; however, there was also a modest relationship in the aorta suggesting for the first time that Klotho may have direct effects on the vasculature. To test this idea further, the authors examined the phenotype of VSMCs in the vessel wall of animals with CKD and high, normal, or low levels of Klotho. Animals lacking Klotho showed upregulated expression of the phosphate transporters Pit1/2 and the key osteogenic transcription factor Runx2. This suggested that in the absence of Klotho, upregulation of Pit1/2 increases Pi transport into VSMCs and this drives osteogenic conversion, a key event in the calcification process.10 However, this observation alone is not strong evidence for direct effects of Klotho on VSMC calcification. Calcification itself can drive VSMC osteogenic differentiation; therefore, such differences may be attributable to different levels of aortic Ca deposition in the different animal models.13,14 More convincing evidence was provided by in vitro studies in which the addition of recombinant soluble Klotho protein to VSMCs was able to decrease high Pi–induced calcification. This inhibition was mediated through suppression of Na-dependent Pi transport into VSMCs, potentially through normalization of mRNA levels encoding Pit1/2. Although tantalizing, this article poses more questions than it answers. First, it does not address how Klotho mediates its effect on VSMCs. Klotho exists in two forms that have