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. 2020 Apr 15;10(2):95–105.

Development of zirconium-89 PET for in vivo imaging of alpha-klotho

Wei Ling Lau 1,*, Christopher Liang 2,*, Han Liu 1, Karanveer Singh 2, Jogeshwar Mukherjee 2
PMCID: PMC7218695  PMID: 32419978

Abstract

Alpha-klotho is a single-pass membrane protein primarily expressed by the kidneys. Klotho deficiency in chronic kidney disease contributes to an accelerated aging phenotype. We report here development of [89Zr]DFO-anti-klotho positron emission tomography (PET) imaging as a novel non-invasive method for assessing whole-body alpha-klotho distribution. Rat monoclonal anti-mouse klotho antibody was reacted with SCN-Bn-deferoxamine (DFO) and was radiolabeled using Zirconium-89. In vitro testing of [89Zr]DFO-anti-mKlotho was done in a distal convoluted tubule kidney cell line and with 40-micron whole kidney sections from C57BL/6J mice. Competitive binding was assessed in co-incubation studies with unlabeled anti-mKlotho antibody. For in vivo testing, C57BL/6J mice were injected retro-orbitally with [89Zr]DFO-anti-mKlotho and were scanned using Inveon PET/CT. Autoradiographs of kidney sections were obtained post-imaging on select animals. Radiochemical yield of [89Zr]DFO-anti-mKlotho was >70% and radiochemical purity was confirmed by iTLC. Specific binding in the kidney cell line was reduced by 60% in the presence of unlabeled anti-mKlotho. In the PET/CT scans, initial uptake of [89Zr]DFO-anti-mKlotho was observed in the intestines and liver. Selective retention of radioactivity was observed in the kidneys in the subsequent 24, 48, and 72 hrs scans with cortical binding of [89Zr]DFO-anti-mKlotho clearly visualized. Sites of lower alpha-klotho expression were not visualized. In summary, we have successfully synthesized [89Zr]DFO-anti-mKlotho and our initial in vitro and in vivo studies in mice demonstrate selective binding in the kidney cortex, which is known to express high levels of alpha-klotho. PET imaging promises to be a novel tool for in vivo evaluation of alpha-klotho distribution.

Keywords: [89Zr]DFO-anti-mKlotho, alpha-klotho, monoclonal antibodies, autoradiography, PET/CT imaging

Introduction

Alpha-klotho was first discovered in 1997 [1] and was named after the Greek goddess Clotho who spins the thread of life because of the molecule’s important role in regulating metabolism and longevity. Alpha- and beta-klotho may play a crucial role in the pathophysiology of aging-related disorders such as diabetes, cancer, arteriosclerosis, and chronic kidney disease [2]. Alpha-klotho is a 130 kDa transmembrane protein with 80% homology between rodents and humans, and functions as the co-receptor for fibroblast growth factor-23 (FGF23) to facilitate FGF23 binding to FGF receptors. FGF23-klotho signaling induces kidney phosphorus excretion and inhibits activity of vitamin D 1α-hydroxylase. A decrease in alpha-klotho is associated with infertility, secondary hyperthyroidism, vascular calcification, cardiac hypertrophy, progression of chronic kidney disease, skin atrophy and osteoporosis [1,3].

Expression of alpha-klotho is primarily in the distal convoluted tubules of the kidney cortex [4]. Low-level expression of alpha-klotho is present in the choroid plexus of the brain and the parathyroid gland. There is controversy surrounding tissue expression of alpha-klotho with discrepancies between animal and human studies, especially in regard to the cardiovascular system [4-6]. Non-invasive methods to examine tissue distribution of alpha-klotho protein in vivo are currently lacking; we propose that development of alpha-klotho imaging would facilitate ongoing research exploring induction of alpha-klotho as a therapeutic target in aging and kidney disease (e.g., novel small chemical compounds and pharmaceutical agents such as active vitamin D and peroxisome proliferator-activated receptor-γ (PPARγ) [2,7]).

Our goal for this project was to develop a positron emission tomography (PET) imaging tool to facilitate in vivo detection of alpha-klotho. Zirconium-89 (89Zr) was chosen as a suitable radioisotope for this study since it possesses a long half-life and has been extensively studied for radiolabeled antibody imaging studies in humans, primary in oncology. For example, 89Zr-labeled antibodies have been used in high-resolution targeted PET-computed tomography (PET/CT) detection of breast cancer [8-12], multiple myeloma cell imaging [13], ovarian cancer [14-17], prostate cancer [18-20], renal and squamous cell carcinoma [21-25] and colon cancer [26-28]. We applied published methods to develop 89Zr radiolabeling of rat monoclonal anti-mouse klotho antibody and herein describe in vitro characterization of the 89Zr-mKlotho conjugate as well as in vivo PET/CT scans in live C57BL/6J mice.

Materials and methods

General methods

All commercially available reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO) unless specified and used without any further purification. p-SCN-Deferoxamine was purchased from Macrocyclics (Plano, TX). Zr-89 in 1 M oxalic acid was produced by 3D Imaging LLC (Little Rock, AR). Rat anti-mouse klotho monoclonal antibody MAB1819 was purchased from R&D Systems (Minneapolis, MN). Water was purified using a MilliQ filtration system. Bio-Rad 6 spin columns were purchased from Bio-Rad (Hercules, CA). PD-10 filtration columns were purchased from GE Healthcare (Chicago, IL). Mass spectrometry was performed using a ESI LC-ToF Micromass LCT Premier (Waters Co., Milford, MA) and Xevo G2-XS ToF-MS (Waters). Analysis of mass spectrometry results was done using MassLynx 4.0 (Waters). Instant thin layer chromatography (iTLC) was performed using chromatography stripes purchased from Biodex and scanned on an AR-2000 Radio-TLC Imaging Scanner (Eckart & Ziegler, Berlin, Germany). Mouse kidney slices were obtained on a Leica 1850 cryotome. Zirconium-89 autoradiographic studies were carried out by exposing tissue samples on storage phosphor screens (Multisensitive Medium MS, PerkinElmer, Waltham, MA). The apposed phosphor screens were read and analyzed by OptiQuant acquisition and the Cyclone Storage Phosphor System (Packard Instruments Co., Boston, MA). A preclinical Inveon dedicated PET scanner (Siemens Medical Solutions, Knoxville, TN) with a transaxial FWHM of 1.46 mm, and axial FWHM of 1.15 mm [29] was used for the PET studies. PET/CT images of mice were obtained and analyzed using ASIProVM and IRW software platforms (Siemens). All animal studies were approved by the Institutional Animal Care and Use Committee of University of California-Irvine.

Synthesis of DFO-anti-mKlotho

The synthesis and radiolabeling of our compound was adapted from a previously published protocol [30]. Anti-mKlotho antibody (500 μg) was dissolved in 0.5 mL sterile saline to create a 0.5 mg/0.5 mL solution of antibody. 450 μL of the resulting solution was added to an Eppendorf tube and 0.1 mL of 0.48 M Na2CO3 was added to raise the solution pH to 9. The solution was vortexed briefly and allowed to rest for 5 minutes. 9 mg DFO was dissolved in 1 mL DMSO; purity of the DFO was confirmed using mass spectrometry. 20 μL of the DFO solution was added to the anti-mKlotho antibody solution in 5 μL aliquots. The mixture was incubated at 37°C for 30 minutes while being gently shaken (Figure 1). The solution was loaded in 100 μL batches into 6 Bio-Rad 6 spin columns and centrifugation was done, discarding the elution. Following first centrifugation, 100 μL saline was added to each Bio-Rad 6 spin column and DFO-anti-mKlotho product was collected in the second elution.

Figure 1.

Figure 1

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Synthesis of [89Zr]DFO-anti-mKlotho. 1. Chemical structure of DFO. 2. Chemical structure of DFO-anti-mKlotho. 3. Chemical structure of [89Zr]DFO-anti-mKlotho.

To confirm successful synthesis of DFO-anti-mKlotho, a 20 μg sample was first treated with dithiothreitol (DTT) to expose the fragments of the antibody. The sample was then analyzed alongside of a pure sample of unconjugated anti-mKlotho antibody using a Xevo G2-XS ToF-MS. DTT-treated pure mKlotho antibody yielded a weight of 24,085 daltons for the klotho fragment. DTT-treated DFO-mKlotho showed both an unlabeled klotho fragment (24,085 daltons) as well as a modified DFO-mKlotho fragment, which was 24,837 daltons (Figure 2). Since the weight of DFO is 752 and 24,085+752 = 24,837, we thus confirmed that our reaction was successful and we had modified the mKlotho antibody with the chelator DFO.

Figure 2.

Figure 2

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Characterization of DFO-mKlotho fragment on mass spectrometry. DTT-treated pure mKlotho antibody yielded a weight of 24,085 daltons for the klotho fragment. DTT-treated DFO-mKlotho showed both an unlabeled klotho fragment (24,085 daltons) as well as a modified DFO-mKlotho fragment, which was 24,837 daltons. Since the weight of DFO is 752 and 24,085+752 = 24,837, we thus confirmed that our reaction was successful and we had modified the mKlotho antibody with the chelator DFO.

Radiosynthesis of [89Zr]DFO-anti-mKlotho

92.13 MBq of [89Zr]oxalate was added to a reaction vial containing 200 μL 1 M oxalic acid. 90 μL of 2 M Na2CO3 was then added and the reaction mixture was allowed to sit for 3 min. HEPES buffer (pH 7.1-7.3; 0.7 mL) was then added followed by the pooled DFO-anti-mKlotho product. The mixture was incubated for one hour at R.T. with gentle shaking (Figure 1).

Afterward, the mixture was purified on a PD-10 column that had been prewashed with gentisic acid (5 mg/mL in 0.25 M NaOAc) and eluted using gentisic acid to collect [89Zr]DFO-anti-mKlotho. A 1.5 mL waste fraction was discarded before collecting the first 2 mL fraction. Afterward, nine more 1 mL fractions were eluted from the PD-10 column. The fractions with the highest radioactivity counts were used for in vivo experiments. To confirm successful radiolabeling of DFO-anti-mKlotho with 89Zr, a sample from one of the selected fractions was used to dot an iTLC chromatography strip that was developed and scanned on a AR-2000 Radio-TLC Imaging Scanner (Eckart & Ziegler). Apparent specific activity of [89Zr]DFO-anti-mKlotho was calculated to be around 0.185 MBq/μg.

In vitro binding assays

In vitro testing of [89Zr]DFO-anti-mKlotho was done with kidney cell lines and 40-micron cryotome sections of mouse whole kidney. Competitive binding was assessed using unlabeled anti-mKlotho antibody. Kidney distal convoluted cells (catalog# ATCC CRL-3250, American Type Culture Collection, Manassas, VA) were expanded and 12 microfuge tubes containing 6.8 million cells each were incubated with 703 MBq [89Zr]DFO-anti-mKlotho per tube. For nonspecific binding, anti-mKlotho was also added to 6 of the tubes (5 μg/mL); final volume was 0.5 mL per sample. Following a 1 hour incubation, the samples were filtered and the microfuge tubes were exposed on phosphor screens.

In vivo mouse PET/CT imaging

Adult male and female C57BL/6J mice were obtained from Jackson Laboratory (Bar Harbor, ME). Under isoflurane anesthesia, [89Zr]DFO-anti-mKlotho 0.925-1.369 MBq was given intravenously via retro-orbital injection. A blocking study was attempted using 10 mg of unlabeled anti-mKlotho that was injected 1 hour prior to the [89Zr]DFO-anti-mKlotho dose.

Scanning was done on the mice at 2, 24, 48 and 72 hr after injection of [89Zr]DFO-anti-mKlotho. The mice were anesthetized using 4% isoflurane gas and then positioned in the mouse chamber of the Inveon Multimodality CT/PET scanner. Anesthesia was maintained with 2-2.5% isoflurane during the scans. Whole body scans were performed using Inveon Multimodality (MM) CT scanner and Inveon Dedicated PET scanner (Siemens). CT images of the mice were obtained with a large area detector (4096 × 4096 pixels, 10 cm × 10 cm field-of-view). The CT projections were acquired with the detector-source assembly rotating over 360 degrees and 720 rotation steps. The mice underwent a single-bed low resolution CT scan at 80 kV/500 uA and a 30-minute PET scan under isoflurane anesthesia for a total scan time of 35-40 min. PET images were reconstructed using the OSEM3D protocol.

Ex vivo studies

After the PET/CT scan, the mice under anesthesia were sacrificed by cardiac exanguination and the kidneys were rapidly removed and frozen in dry ice. Kidney sections of 40-micron thickness were generated on a cryotome and mounted on glass slides. The slides were then exposed on storage phosphor screens and analyzed.

Image analysis

The scans were analyzed using Inveon Research Workplace. Regions of interest (ROIs) were first determined by looking at the CT scan and locating the kidneys. Separate ROIs were drawn around the kidney cortex and medulla with care taken to avoid overlap of the two ROIs. ROIs were constructed to be approximately the same size. The mean uptake of the respective regions was recorded in Bq/mL. Standardized uptake values (SUVs) were calculated using recorded weights and injection doses. Screenshots were obtained using the in-program screenshot option. 3D images were generated using the Inveon Research Workplace 3D Visualization program.

Results

Synthesis of DFO-anti-mKlotho

We achieved successful synthesis of DFO-anti-mKlotho consistent with previously reported methods [30] (Figure 1). We did require one modification whereby the spin columns had to be centrifuged twice in order to elute the DFO-anti-mKlotho product. It remains unclear why the elution rate was slower compared to published methods, however mass spectrometry confirmed successful synthesis of DFO-anti-mKlotho despite the delayed elution time.

Radiosynthesis of [89Zr]DFO-anti-mKlotho

Synthesis of [89Zr]DFO-anti-mKlotho was successfully carried out published Zr-89 methods [30] with a radiochemical yield of greater than 70%. The majority of the [89Zr]DFO-anti-mKlotho eluted within 6-8 fractions (Figure 3A). RadioTLC determined that radiochemical purity was greater than 90%; aqueous citrate eluent, Rf<0.1 (Figure 3B). Apparent specific activity of [89Zr]DFO-anti-mKlotho was calculated to be approximately 0.185 MBq/μg.

Figure 3.

Figure 3

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Radiosynthesis results and purity analysis. A. The amount of [89Zr]DFO-anti-mKlotho activity obtained in each fraction of the PD-10 elution. B. RadioTLC of the completed product confirming that the majority of the eluted product stays within region 1 of the iTLC strip, consistent with successful radiolabeling.

In vitro binding affinity studies

As expected, [89Zr]DFO-anti-mKlotho showed significant binding to cultured distal convoluted tubule kidney cells which are known to express alpha-klotho [4]. In the presence of unlabeled anti-mKlotho antibody, specific binding was reduced by 60% with signal from the radiolabeled compound reduced to 30-40% (Figure 4).

Figure 4.

Figure 4

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Autoradiograph of distal convoluted tubule kidney cell line featuring total binding and non-specific binding. A. Total binding of [89Zr]DFO-anti-mKlotho. B. Non-specific binding measured in the presence of 5 mg/mL of anti-mKlotho. C. The chart summarizes radioactive uptake (digital light units, DLU/mm2) of the [89Zr]DFO-anti-mKlotho with calculated total, non-specific and specific binding values. The specific binding of [89Zr]DFO-anti-mKlotho in the cultured kidney cell line, derived from blocking studies with unlabeled anti-mKlotho antibody, was 30-40%.

In vitro incubation with the cryo-preserved kidney sections showed that the majority of [89Zr]DFO-anti-mKlotho binding was within the kidney cortex with lesser binding detected in the medulla (Figure 5). The ratio of cortex:medulla signal was calculated at 1.25. When co-incubated with unlabeled anti-mKlotho antibody, nonspecific binding was high indicating successful blocking of alpha-klotho antigen sites.

Figure 5.

Figure 5

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In vitro [89Zr]DFO-anti-mKlotho binding in kidney sections. (A) Cryopreserved mouse kidney sections (40-micron thickness) were incubated with [89Zr]DFO-anti-mKlotho. (B) Total binding of [89Zr]DFO-anti-mKlotho. (C) Non-specific binding where sections were pre-incubated with unlabeled anti-mKlotho (1.5 mg/ml). (D) Plot showing total and nonspecific binding of [89Zr]DFO-anti-mKlotho in (B and C). There was greater binding within the cortex compared to the medulla with a ratio of 1.25. Ratio of total cortex to nonspecific cortex was 1.32.

In vivo PET/CT scans

Two male and 5 female mice completed in vivo imaging. Due to initial difficulties with tracer injections leading to poor tissue signal, we had limited imaging data from male animals. PET/CT scans showed initial uptake of [89Zr]DFO-anti-mKlotho in the intestines and liver, followed by persistent distinct binding of [89Zr]DFO-anti-mKlotho in the kidneys within 24 hours of injection. Localization of binding was confirmed using the co-registered CT scan. The majority of [89Zr]DFO-anti-mKlotho signal was in the kidney cortex, with lower intensity in the kidney medulla (Figure 6). Serial PET/CT scans showed mild decrease in [89Zr]DFO-anti-mKlotho activity within the kidneys due to radioactive decay at 48 and 72 hours after injection. Binding was symmetric in both kidneys and the ratio of cortex:medulla uptake was 1.32. The liver had detectable but much lower uptake of the radiolabeled antibody. The PET scans did not demonstrate [89Zr]DFO-anti-mKlotho binding in other organs. In one mouse with 10 μg unlabeled anti-mKlotho antibody injected 1 hour prior to [89Zr]DFO-anti-mKlotho (blocking study), no significant reduction in the binding of [89Zr]DFO-anti-mKlotho was observed. This may be due to the low dose of unlabeled anti-mKlotho antibody (limited by cost) and/or a general difficulty in carrying out displacement/blocking studies with antibodies.

Figure 6.

Figure 6

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Sequential 30-minute static scans of [89Zr]DFO-anti-mKlotho distribution. PET/CT imaging was done at (A) 2 hours, (B) 24 hours, and (C) 48 hours after retro-orbital injection of [89Zr]DFO-anti-mKlotho. (D) Standardized uptake values (SUVs) summarized in the graph show optimal uptake of the radiolabeled antibody in the kidneys at 24 hours, with slight decay of radioactivity after 48 hours. The ratio of cortex:medulla signal was 1.32 at 24 hours and 1.49 at 48 hours. (E) Axial (left panel), coronal (middle panel) and sagittal (right panel) images taken 72 hours after injection of [89Zr]DFO-anti-mKlotho in a female mouse, demonstrating persistent strong radioactivity in the kidneys. A region of activity in the liver was also noted (yellow arrow).

Ex vivo studies

Kidneys were harvested 72 hours after retro-orbital injection of [89Zr]DFO-anti-mKlotho, following completion of PET/CT scans. Cryo-preserved ex vivo kidney sections exposed on phosphor screens demonstrated localized binding in the kidney cortex with minimal uptake in the medulla (Figure 7). The ratio of uptake in the cortex versus medulla in the female mouse was 3.74 versus a lower cortex:medulla ratio of 2.72 in the male mouse.

Figure 7.

Figure 7

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Ex vivo images of kidney sections harvested from mice after PET/CT imaging. Imaging of the kidneys harvested from mice 72 hours after retro-orbital injection of [89Zr]DFO-anti-mKlotho showed more robust cortex:medulla selective activity than the in vitro studies. A. Female mouse kidney section showing distinct binding in the cortex. B. Male mouse kidney section showing lower binding in the cortex compared to the female. C. Plot shows results from A and B cortex and medulla binding of [89Zr]DFO-anti-mKlotho. The ratio of kidney cortex binding compared to medulla was 2.72 in male animals and 3.74 in female animals.

Discussion

In vivo intravenous administration of [89Zr]DFO-anti-mKlotho demonstrated localized binding in the kidneys, consistent with the kidneys being the primary source of alpha-klotho. There was higher binding in the cortex than the medulla of the kidneys and this signal was attenuated in blocking studies with unlabeled anti-mKlotho antibody. In vitro binding with a distal convoluted tubule cell line and kidney sections showed similar results with blocking of [89Zr]DFO-anti-mKlotho by unlabeled antibody; however in vitro studies were limited by low specific binding.

The discrepancy between the strong in vivo PET/CT kidney cortex signal (confirmed on ex vivo kidney sections) and the low in vitro specific binding (in cultured cells and kidney sections) is likely due to alpha-klotho trafficking across renal tubules that happens in vivo. Hu et al. previously demonstrated that transcytosis of alpha-klotho from the blood circulation occurs from basolateral to intracellular location, with subsequent secretion across the apical membrane in both proximal and distal tubules [31]. The selective targeting of the kidneys by labeled [89Zr]DFO-anti-mKlotho that was persistent out to 72 hours post-injection was unlikely to be due to nonspecific glomerular filtration, since previous studies reported that [89Zr]DFO clears out quickly from the urinary system within an hour after injection and shows only minimal retention in the kidneys [30,32]. There was little background noise in the PET scans outside of the liver, which had marginal uptake of the antibody. This could be due to continued processing of [89Zr]DFO-anti-mKlotho through the bloodstream, as there is no reported literature of [89Zr]DFO being trapped within the liver.

We were able to detect localization of 89Zr-labeled alpha-Klotho antibody in the kidney, which is likely to have concentrations greater than 500 pg/ml based on extrapolation from serum alpha-klotho data [2]. While prior in vitro investigations have shown that there are alpha-klotho production sites within the choroid plexus and the parathyroid gland [4], there was no discernible [89Zr]DFO-anti-mKlotho binding to these sites in vivo in the current study. This may be due to the blood-brain barrier being impermeable to the complexed agent, preventing access to the choroid plexus. With regard to the parathyroid gland, it is possible that [89Zr]DFO-anti-mKlotho concentration in vivo was not high enough to produce a detectable signal. Further, it has been proposed that extra-renal alpha-klotho production may be low or undetectable under normal alpha-klotho status in the setting of intact kidney function [31].

The ev vivo results with the kidney slices showed a significant difference in uptake between the kidney cortex and the kidney medulla, with 2.7-3.7 times higher uptake in the cortex than medulla (Figure 7). These results are the most significant finding to come out of our experiments, since the kidney cortex is the major source of alpha-klotho production within the body. Female mice showed more robust cortex:medulla binding than male animals; the physiologic relevance of this is unclear at this time. Sex- and age-dependent modulation of alpha-klotho has been reported in humans; serum alpha-klotho levels are higher in young girls compared to boys, whereas cerebrospinal fluid alpha-klotho is lower in females in the geriatric population [33,34].

In summary, [89Zr]DFO-anti-mKlotho is a novel radiolabeled antibody that can be used for selective imaging of alpha-klotho protein in vivo. The synthesis protocol resulted in high radiochemical purity and yield. There is little degradation of the DFO complex in vivo, providing a clear picture of the compound’s biodistribution. The radiolabeled antibody travels selectively to the kidneys, with minor binding located in the liver. In vivo scans and ex vivo autoradiographs show intense uptake within the kidney cortex compared to the medulla, which is consistent with previous in vitro studies of alpha-klotho. In future studies we plan to utilize [89Zr]DFO-anti-mKlotho PET/CT imaging to examine alpha-klotho biodistribution in models of alpha-klotho deficiency such as chronic kidney disease and aging.

Acknowledgements

This work was supported by an American Heart Association Innovative Research Grant 17IRG33410803 (WLL). We thank the UCI Undergraduate Research Opportunities Program for support (KS), and acknowledge valuable input from Anna Wu (Radiation Oncology, City of Hope, Los Angeles, CA), Felix Bergara (Molecular and Medical Pharmacology University of California-Los Angeles), Ming Chang Hu and Orson Moe (Nephrology/Internal Medicine, UT Southwestern, Dallas, TX) during the design of the experiments. Pilot findings from this project were presented in an oral abstract at the 2019 Society of Nuclear Medicine and Molecular Imaging (SNMMI) Annual Meeting (Anaheim, CA, June 2019).

Disclosure of conflict of interest

None.

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