Abstract
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Objectives: Alpha-Klotho is a single-pass membrane protein primarily expressed by the kidneys. Klotho deficiency in chronic kidney disease (CKD) has been implicated in accelerated aging. Non-invasive methods for assessing whole-body Klotho tissue distribution are currently unavailable. We report here radiosynthesis and biological evaluation of [89Zr]DFO-anti-Klotho as a potential PET imaging agent for Klotho.
Methods: Rat monoclonal anti-mouse Klotho antibody was dissolved in sterile saline (1 μg/μL) and 0.1 M Na2CO3 was added to raise the pH to 9. To this 120 μg in 20 μL DMSO of p-SCN-Bn-Deferoxamine (Macrocyclics, Inc.) was added in 5 μL portions and reacted at 37oC for 30 minutes. The anti-mKlotho-DFO mixture was purified using Bio-Rad Bio-Spin 6 Tris columns. For radiolabeling, 2 to 4 mCi of [89Zr]oxalate (3D Imaging LLC, Inc.) in 200 μL 1 M oxalic acid was treated with 90 μL 2 M Na2CO3 for 3 min. To this [89Zr]oxalate solution, HEPES buffer was added followed by anti-mKlotho-DFO. Additional HEPES was added and incubated for one hour at R.T. while gently shaking. The mixture was purified on a prewashed PD-10 column and eluted using gentisic acid solution to collect pure [89Zr]DFO-anti-mKlotho. In vitro testing of [89Zr]DFO-anti-mKlotho was done in a distal convoluted tubule kidney cell line and 40-micron whole kidney sections from normal and chronic kidney disease (CKD) mice. Competitive binding was assessed using unlabeled anti-mKlotho. For in vivo testing, C57BL/6J mice were injected with [89Zr]DFO-anti-mKlotho (10-30 μCi retro-orbitally) and were scanned using Inveon PET/CT between the 2 and 72 hrs. Autoradiographs of kidney sections were obtained post-PET/CT on select animals. Optiquant for autoradiographs and Inveon Research Workplace (IRW) for PET/CT image analysis were used.
Results: Radiochemical yield of [89Zr]DFO-anti-mKlotho was >70% and radiochemical purity was confirmed by ITLC (aqueous citrate eluent, Rf<0.1). Specific binding in the kidney cell line was reduced by 60% in the presence of unlabeled anti-mKlotho. Kidney sections from CKD vs control animals showed a 34% reduction of cortex binding (adjusted for medulla background signal). In the presence of unlabeled anti-mKlotho a 27% reduction was observed in controls. In the PET/CT scans, after initial uptake of [89Zr]DFO-anti-mKlotho in the intestines, gut and liver selective retention of radioactivity was observed in the kidneys in the subsequent 24, 48, and 72 hrs scans with cortex binding of [89Zr]DFO-anti-mKlotho clearly visualized. PET/CT image below is of a control mouse injected with [89Zr]DFO-anti-mKlotho. The 30 minute static scan was taken 48 hours after injection and localized greater uptake in the bilateral kidney cortex is observed. Ex vivo kidney autoradiographs after PET/CT scans showed a 38% reduction of kidney cortex/medulla signal in CKD vs control animals.
Conclusions: We have successfully synthesized [89Zr]DFO-anti-mKlotho and our initial in vitro and in vivo studies in mice suggest selective binding in the kidney cortex, which is known to express high levels of Klotho. Further confirmatory in vivo blocking and CKD mouse studies are in progress. PET imaging of Klotho promises to be a powerful tool to evaluate diseases with altered Klotho expression such as CKD. RESEARCH SUPPORT: AHA 171RG33410803 (WLL)