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