Establishment of sandwich ELISA for soluble alpha-Klotho measurement: Age-dependent change of soluble alpha-Klotho levels in healthy subjects

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Abstract

Background

α-Klotho (αKl) regulates mineral metabolism such as calcium ion (Ca2+) and inorganic phosphate (Pi) in circulation. Defects in mice result in clinical features resembling disorders found in human aging. Although the importance of transmembrane-type αKl has been demonstrated, less is known regarding the physiological importance of soluble-type αKl (sαKl) in circulation.

Objectives

The aims of this study were: (1) to establish a sandwich ELISA system enabling detection of circulating serum sαKl, and (2) to determine reference values for sαKl serum levels and relationship to indices of renal function, mineral metabolism, age and sex in healthy subjects.

Results

We successively developed an ELISA to measure serum sαKl in healthy volunteers (n = 142, males 66) of ages (61.1 ± 18.5 year). The levels (mean ± SD) in these healthy control adults were as follows: total calcium (Ca; 9.46 ± 0.41 mg/dL), Pi (3.63 ± 0.51 mg/dL), blood urea nitrogen (BUN; 15.7 ± 4.3 mg/dL), creatinine (Cre; 0.69 ± 0.14 mg/dL), 1,25 dihydroxyvitamin D (1,25(OH)2D; 54.8 ± 17.7 pg/mL), intact parathyroid hormone (iPTH; 49.2 ± 20.6 pg/mL), calcitonin (26.0 ± 12.3 pg/mL) and intact fibroblast growth factor (FGF23; 43.8 ± 17.6 pg/mL).

Serum levels of sαKl ranged from 239 to 1266 pg/mL (mean ± SD; 562 ± 146 pg/mL) in normal adults. Although sαKl levels were not modified by gender or indices of mineral metabolism, sαKl levels were inversely related to Cre and age. However, sαKl levels in normal children (n = 39, males 23, mean ± SD; 7.1 ± 4.8 years) were significantly higher (mean ± SD; 952 ± 282 pg/mL) than those in adults (mean ± SD; 562 ± 146, P < 0.001). A multivariate linear regression analysis including children and adults in this study demonstrated that sαKl correlated negatively with age and Ca, and positively with Pi. Finally, we measured a serum sαKl from a patient with severe tumoral calcinosis derived from a homozygous missense mutation of α-klotho gene. In this patient, sαKl level was notably lower than those of age-matched controls.

Conclusion

We established a detection system to measure human serum sαKl for the first time. Age, Ca and Pi seem to influence serum sαKl levels in a normal population. This detection system should be an excellent tool for investigating sαKl functions in mineral metabolism.

Research highlights

► We established a detection system to measure human serum soluble α-Klotho (sαKl). ► We found that Age influences serum sαKl levels in a normal population. ► This detection system should be an excellent tool for investigating sαKl functions.

Introduction

α-kl Gene encodes a type I membrane protein with expression restricted to parathyroid glands, the choroid plexus and the kidney [1], [2], [3], [4]. αKl binds to Na+,K+-ATPase to regulate PTH secretion and is involved in transepitherial calcium concentration. In response to altered extra-cellular calcium concentrations, αKl is rapidly translocated from endosomal organella to the plasma membrane together with Na+,K+-ATPase and simultaneously the extracellular domain of αKl is cleaved and secreted into the blood circulation and cerebrospinal fluid (CSF) [5], [6]. The increased Na+ gradient produced by elevated Na+,K+-ATPase activity drives PTH secretion in parathyroid glands and transepithelial transport of calcium in the kidney and choroid plexus [5]. Accordingly, it is assumed that αKl levels in the serum and CSF mirror the molecular actions of the cellular form of αKl in these tissues.

αKl also binds to fibroblast growth factor 23 (FGF23), which was discovered in studies of autosomal dominant hypophosphatemic rickets (ADHR) [7] and later tumor-induced osteomalacia (TIO) [8], [9]. FGF23 (i) is produced and secreted from bone in response to serum levels of phosphorus and 1,25(OH)2D [10], [11], [12], (ii) binds to FGF receptor1 (FGFR1), both suppressing 1α-hydroxylase (CYP27B1) expression and stimulating 24-hydroxylase (CYP24A1) expression in kidney [13], [14], and (iii) downregulates protein amounts of Na+ dependent-phosphate transporter (NaPi) IIa/c to the brush border membrane of proximal tubules thus decreasing phosphate reabsorption [11].

αKl contributes to integrate mineral homeostasis. Consequently, disturbances of αKl expression impair mineral metabolism via multiple mechanisms involving FGF23 signaling [13], [14], PTH secretion and transepithelial calcium transport. Recently, a patient with autosomal recessive hyperphosphatemic tumoral calcinosis shed new light on the impact of αKl [15]. Mutation analysis revealed a missense mutation in αKl, and in vitro studies indicated that αKl translocation to the plasma membrane was impaired [16]. Therefore, analysis of serum αKl levels may lead to greater understanding of disorders of mineral homeostasis.

In the present study, we developed an ELISA system to measure circulating sαKl concentrations in serum from human subjects for the first time. We further analyzed and compared sαKl levels of both healthy volunteers and a case with the α-klotho gene mutation [16]. Finally, we discuss the potential utility in measuring serum αKl in clinical disorders.

Section snippets

Plasmid construction

Human full length α-Klotho (fl-αKl; 1012 amino acids(a.a), RefSeq ID: NP_004786)-cDNA and cDNA encoding extracellular domain of α-Klotho (sαKl; 1-979a.a.) were amplified from total human kidney cDNAs by PCR and consequently cloned into pLP-CMVneo and pLP-IRESneo, respectively, by In fusion PCR kit (Clonetech).

Cell culture

pLP-CMVneo-fl-αKl was transfected into HEK293 cells by the calcium-phosphate method. pLP-IRESneo-sαKl was transfected into CHO cells, by the Lipofectamine method (Invitrogen). Then, cells

Establishment of anti-αKl mouse monoclonal antibodies for sandwich ELISA

To establish mouse monoclonal antibodies with strong affinity for human αKl protein, we immunized mice with a human full-length αKl expressing vector and screened the resulting hybridomas by measuring the binding affinities to human αKl expressing cells. Nine antibodies which showed high and specific affinities to an extracellular domain of human αKl protein were tested for compatibility as a capture antibody or detection antibody in sandwich ELISA to detect sαKl protein. We finally selected a

Discussion

We developed an ELISA to successfully measure sαKl in human subjects and demonstrated positive relationships with phosphorus, and negative relationships with age and Ca2+ in multiple linear regression analysis. Since low calcium stimuli induce αKl secretion [5], the negative relationship appears compatible. We have previously proposed a novel negative feedback system involving FGF23 and αKl for maintenance of normal phosphate and 1,25(OH)2D levels [16], [13]. Based on the fact that FGF23 is a

Acknowledgments

This work was supported in part by the grants from Ministry of Education, Science and Culture in Japan, 21026017, 21390058 (to A.I.) and 17109004 (to Y.N.) and the Ministry of Health, Labour and Welfare of Japan KH20Q007a-1 (to K.O.). We thank K. Sakuma, K. Ono and N. Yoshii for excellent technical support and Dr. T Sakai for helpful suggestion. We thank the coordinators of Fujiwara-kyo Study [20] from which almost healthy adults were recruited. Notably, subjects from Fujiwara-kyo Study had no

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