The demonstration of αKlotho deficiency in human chronic kidney disease with a novel synthetic antibody

Abstract

Background: αKlotho is the prototypic member of the Klotho family and is most highly expressed in the kidney. αKlotho has pleiotropic biologic effects, and in the kidney, its actions include regulation of ion transport, cytoprotection, anti-oxidation and anti-fibrosis. In rodent models of chronic kidney disease (CKD), αKlotho deficiency has been shown to be an early biomarker as well as a pathogenic factor. The database for αKlotho in human CKD remains controversial even after years of study.

Methods: We used a synthetic antibody library to identify a high-affinity human antigen-binding fragment that recognizes human, rat and mouse αKlotho primarily in its native, rather than denatured, form.

Results: Using an immunoprecipitation-immunoblot (IP-IB) assay, we measured both serum and urinary levels of full-length soluble αKlotho in humans and established that human CKD is associated with αKlotho deficiency in serum and urine. αKlotho levels were detectably lower in early CKD preceding disturbances in other parameters of mineral metabolism and progressively declined with CKD stages. We also found that exogenously added αKlotho is inherently unstable in the CKD milieu suggesting that decreased production may not be the sole reason for αKlotho deficiency.

Conclusion: Synthetic antibody libraries harbor tremendous potential for a variety of biomedical and clinical applications. Using such a reagent, we furnish data in support of αKlotho deficiency in human CKD, and we set the foundation for the development of diagnostic and therapeutic applications of anti-αKlotho antibodies.

Keywords: assay; chronic kidney disease; klotho; synthetic antibodies.

FIGURE 1:

Sequence, specificity and affinity of sb106 (A) Sequences of the CDRs of sb106 in the IMGT numbering scheme [69]. (B) Specificity of sb106 by phage ELISA. Sb106 phage were assayed for binding to the following immobilized proteins: human αKlotho (Hu αKL), mouse αKlotho (Mu αKL), complex of the extracellular domain of mouse αKlotho and the ligand-binding domain of human FGFR1c (Mu αKL:FGFR1c), ligand-binding domain of human FGFR1c (FGFR1c), neutravidin (NAV), and bovine serum albumin (BSA). (C) Estimation of the affinity of sb106 for αKlotho by competitive phage ELISA. Sb106 phage were pre-incubated with serial dilutions of human αKlotho (x-axis), prior to capture with immobilized antigen and detection by a colorimetric assay (y-axis).

FIGURE 2:

Characterization of sb106-Fab by immunoblot, immunohistochemistry and immunocytochemistry (A) Immunoblot of kidney lysate from wild-type (WT) mice, homozygous αKlotho hypomorphic mice (kl/kl) and transgenic αKlotho-overexpressing mice (Tg-Kl), using the monoclonal antibody KM2076 or the sb106-Fab. GAPDH: glyceraldehyde phosphate dehydrogenase. (B) Immunoblot of lysates from normal rat kidney (NRK) cells, human embryonic kidney (HEK) cells and HEK cells transfected with a plasmid for over-expression of αKlotho, using the monoclonal antibody KM2076 or the sb106-Fab. (C) Fresh or fixed rat parathyroid tissue probed with phalloidin for β-actin (green) or sb106-IgG (red). (D) HEK293 cells transfected with empty vector or vector for over-expression for αKlotho or βKlotho, stained with sb106-Fab (green) and DAPI (blue).

FIGURE 3:

Characterization of sb106-Fab by immunoprecipitation. (A) HEK293 cells were transfected with empty vector or varying quantities (µg/dish) of vector for expression of transmembrane full-length αKlotho (TM-αKlotho) or soluble extracellular domain of αKlotho with a C-terminal FLAG epitope (s- αKlotho-FLAG). Cell lysates or cell culture medium was immunoprecipitated (IP) with either sb106-Fab or anti-FLAG MAb. Immunocomplexes were resolved by SDS–PAGE and immunoblotted (IB) with monoclonal anti-αKlotho antibody KM2076. (B) Urine from rat, mouse or human was immunoprecipitated with sb106-Fab, resolved by SDS–PAGE and immunoblotted (IB) with KM2076 (left three lanes). Size-selected urine (100-kDa cutoff) was directly subjected to SDS–PAGE and immunoblotted (right three lanes). (C) Sera from a healthy volunteer (healthy), a patient with end-stage renal disease (ESRD), WT mice and homozygous Klotho hypomorphic mice with systemic Klotho deficiency (kl/kl) were subjected to immunoprecipitation by sb106-Fab and immunoblot by KM2076.

FIGURE 4:

Validation of IP-IB assay using human serum spiked with recombinant αKlotho. (A) Known amounts of soluble human αKlotho ectodomain were added to sera from a healthy volunteer or an anuric dialysis patient (CKD patient). αKlotho was measured in the sera using the IP-IB assay. (B) Similar experiment as in (A) except comparisons was made where protease inhibitors (AEBSF 0.1 mm, aprotinin 0.3 µm, bestatin 10 µm, E-64 1 µm, leupeptin 50 µm, pepstatin A 1 µm) were either included or excluded from the IP. (C) αKlotho levels determined by IP-IB (y-axis) were plotted against the added recombinant αKlotho (x-axis) in the four conditions described earlier. Extrapolation to zero spiking shows the level of endogenous αKlotho in the serum treated with protease inhibitors. Only one line is shown for healthy serum with or without protease inhibitors as the results were indistinguishable.

FIGURE 5:

IP-IB assay of serum αKlotho in humans with chronic kidney disease. (A) αKlotho was measured by the IP-IB assay in human sera from normal healthy volunteers and patients from a CKD clinic and dialysis unit using the conventional numerical staging using recombinant αKlotho as a calibration curve. Bars and error bars denote means and standard deviations. The data were analyzed by ANOVA followed by Student–Newman–Keuls test for pairwise multiple comparisons. P-values achieving statistical significance between groups are indicated above the brackets. The number of subjects in each group is indicated at the bottom. (B) The concentrations of αKlotho in a large variety of human sera were determined either by IP-IB (x-axis) or by a commercial ELISA (y-axis) in the same samples. The dotted line represents identity. The black diamonds represent sera that have been through one or more freeze-thaw cycles (stored), and the gray diamonds represent sera thawed only once (fresh). (C) Sera from human subjects were assayed by IP-IB and ELISA. The same sera were subjected to the indicated cycles of repeated freeze-thaw and then assayed. Results for each sample were expressed as a percentage of the reading from the same sample thawed only once. The heavy lines denote the mean of the different subjects.

FIGURE 6:

Human urinary αKlotho levels. αKlotho was measured in the urine of healthy volunteers or patients with chronic kidney disease stage 5 (CKD5). (A) A representative IP-IB assay using recombinant murine αKlotho (rMKl) as a calibration with four subjects in each group under steady state conditions. Equal amounts of urine creatinine were used for IP-IB. (B) Summary of the data from the IP-IB assay and the commercial ELISA. Bars and error bars represent mean and standard deviation from eight subjects in each group. The mean of the healthy volunteers was set as a reference of 100%.