The enzymatic activity of inositol hexakisphosphate kinase controls circulating phosphate in mammals

Circulating phosphate levels are tightly controlled within a narrow range in mammals. By using a novel small-molecule inhibitor, we show that the enzymatic activity of inositol hexakisphosphate kinases (IP6K) is essential for phosphate regulation in vivo. IP6K inhibition suppressed XPR1, a phosphate exporter, thereby decreasing cellular phosphate export, which resulted in increased intracellular ATP levels. The in vivo inhibition of IP6K decreased plasma phosphate levels without inhibiting gut intake or kidney reuptake of phosphate, demonstrating a pivotal role of IP6K-regulated cellular phosphate export on circulating phosphate levels. IP6K inhibition-induced decrease in intracellular inositol pyrophosphate, an enzymatic product of IP6K, was correlated with phosphate changes. Chronic IP6K inhibition alleviated hyperphosphataemia, increased kidney ATP, and improved kidney functions in chronic kidney disease rats. Our results demonstrate that the enzymatic activity of IP6K regulates circulating phosphate and intracellular ATP and suggest that IP6K inhibition is a potential novel treatment strategy against hyperphosphataemia.


Supplementary Methods
Characterisation of SC-919 SC-919, a single isomer, was prepared via stereoselectivity according to our developed method 1 . The absolute configuration of SC-919 was determined using X-ray crystallography; CCDC (Deposition Number) 2075338.

Kinase selectivity assay (kinome-wide selectivity profile of SC-919)
The kinome-wide selectivity profile of SC-919 was analysed using the KINOMEscan technology provided by DiscoveRx 2 . For most of the assays, kinase-tagged T7 phage strains were grown in parallel in 24-well blocks in an E. coli host derived from the BL21 strain. E. coli cells were grown until the log phase and infected with T7 phage from a frozen stock (multiplicity of infection = 0.4) and incubated with shaking at 32 °C until lysis occurred (90-150 min). The lysates were centrifuged (6,000 × g) and filtered (0.2 µm) to remove the cell debris. The remaining kinases were expressed in HEK293 cells and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 min at 22-26 °C to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock [Pierce], 1 % BSA, 0.05 % Tween 20, 1 mM DTT) to remove the unbound ligand and to reduce the non-specific binding of phages. The binding reactions were assembled by mixing the kinases, liganded affinity beads, and test compounds in 1× binding buffer (20 % SeaBlock, 0.17× PBS, 0.05 % Tween 20, 6 mM DTT). Test compounds were prepared as 40× stocks in 100 % DMSO and were directly diluted into the assay mixture. All the reactions were performed in 384-well polypropylene plates at a final volume of 0.02 mL. The assay plates were incubated at 22-26 °C with shaking for 1 h, and the affinity beads were washed with wash buffer (1× PBS, 0.05 % Tween 20). The beads were resuspended in elution buffer (1x PBS, 0.05 % Tween 20, 0.5 µM non-biotinylated affinity ligand) and incubated at 22−26 °C with shaking for 30 min. The concentrations of kinases in the eluates were measured using qPCR. Detailed information is available at the Eurofin DiscoveRx website (https://www.discoverx.com/).

Qualification for the measurement of InsP 6 and InsP 7 Preparation of SC-919-treated sample for quality control (QC) sample
Cells (293, HAP1 wild-type) were treated with the 1 μM of SC-919 for 4 h, and were then immediately treated with trypsin and 10 % FCS-supplemented medium containing similar concentrations of SC-919. The cells were collected and centrifuged (2,000 × g, 1 min) and the supernatant was removed. The animals were used for in vivo experiments when they were 10 weeks old (Sprague-Dawley (SD) rats). SC-919 (10 mg/kg) was administered, and after 6 h, the rats were anesthetised with isoflurane (1-5 % v/v) and sacrificed by exsanguination to collect tissue samples. The cell and tissue samples were placed at −80 °C until required for analysis. The frozen tissue or cell samples were mixed with 3.6 % perchloric acid (v/v, prepared in distilled water [Nippon Gene]) (prepared using 0.2 and 0.3 mL of 3.6 % PCA per cell sample [1 × 10 6 and 4 × 10 6 cells for 293 and HAP1 wild-type cells, respectively] and 100 mg tissue samples). The samples incubated on ice were subsequently immediately homogenised for 2.5-10 min with zirconia beads (5 mm) using a mechanical homogeniser (1,100 rpm; Shakemaster, Bio Medical Science, Japan) followed by a spin-down. Subsequently, one-third (v/v, against PCA) of 30 % (w/v) potassium chloride solution was mixed with the homogenised samples with vortexing.
Next, samples were centrifuged (20,000 × g, 5 min, 4°C) and were used for subsequent analyses in InsP6 and InsP7 measurements.

Method qualification for the determination of InsP6 and InsP7 levels in tissues and cells using LC/MS/MS
LC/MS/MS conditions for determination of InsP6 [099107, Matrix Scientific] and InsP7 are shown in the main text. Stock solutions were prepared by dissolving accurately weighted InsP6 or InsP7 in water to yield a concentration of 10 mg/mL for InsP6, 1 mg/mL for InsP7. These stock solutions were further diluted in water to prepare working solutions for calibration curve, quality control (QC) samples and dilution integrity QC (DIQC) samples. The concentrations of working solutions ranged from 600 ng/mL to 600,000 ng/mL for InsP6, 3 ng/mL to 30,000 ng/mL for InsP7. Rat blank plasma was mixed with 3.6 % perchloric acid (v/v, prepared in distilled water) (prepared using 0.3 mL of 3.6 % PCA per 0.1 mL rat blank plasma). Subsequently, one-third (v/v, against PCA) of 30 % (w/v) potassium chloride solution was mixed with the mixed rat blank plasma with vortexing. Next, the mixtures were centrifuged (20,000 × g, 5 min, 4°C). The supernatant (PCA/potassium chloride-treated plasma) was used as blank matrix for preparation of calibration standard, and rat blank plasma was used as a surrogate matrix for tissues and Cells. Calibration standards for InsP6 or InsP7 were prepared by adding 10 µL of working solution to 150 µL of PCA/potassium chloride-treated plasma. QC samples in SC-919-treated sample for InsP6 or InsP7 were prepared by adding 10 µL of working solution to 150 µL of prepared SC-919-treated cell or tissue samples (see the section of "Preparation of SC-919-treated sample for quality control (QC) sample") at low (LQC), medium (MQC) and high (HQC) levels. Dilution integrity QC samples (DIQC) for InsP7 in kidney were prepared by adding 8 µL of working solution at 30,000 ng/mL to 592 µL of prepared SC-919-treated kidney samples (see section "Preparation of SC-919-treated sample for quality control (QC) sample") at 400 ng/mL. After mixing, the mixtures were diluted with PCA/potassium chloride-treated plasma to 40 ng/mL (diluted DIQC sample). These calibration standards, QC and diluted DIQC samples were spiked with internal standard solution (20 µL, 500 ng/mL InsP6-d6 as an internal standard, 2, 500 ng/mL N, N, N', N' -ethylenediaminetetrakis (methylenephosphonic acid) [E0393, Tokyo Chemical Industry]). After mixing, the mixtures were centrifuged (20,000 × g, 5 min, 4°C ), and the supernatant was subjected to ultrafiltration (15,000 × g, 30 min, 4°C; Amicon Ultra-0.  Fig. 3 and Supplementary Fig. 4 (representative chromatogram of LQC).

Method for Supplementary Fig. 6 Preparation of protein samples for western blot analysis
Wild-type and XPR1 KO HAP1 cells were cultured (200,000 cells/well) in 12-well culture plates with IMDM containing 10 % FCS. As an XPR1-positive control, the human XPR1-expressing vector was introduced into XPR1 KO cells using the Xfect reagent following the procedure described in the Methods section. The culture medium was replaced the next day, and the cells were cultured for one more day. The culture medium was then removed, and the cells were treated with M-PER™ Mammalian Protein Extraction Reagent (500 μL) containing the protease inhibitor cocktail (cOmplete ™ ULTRA) by shaking the plate for 10 min at 22-26 °C. Samples were collected and centrifuged (12,000 × g, 1 min, 4 °C). The supernatant (250 μL) was mixed with 4× Laemmli Sample Buffer (250 μL, Bio-Rad) and 10 M urea solution (500 μL, final 5 M) in 2-mL tubes. The samples were properly mixed for 8 h at 22-26 °C using a rotator and used for western blot analysis. We used 10 (HAP1 wild-type and XPR1 KO cells) and 2 μL (XPR1overexpressed XPR1 KO cells) samples for loading.

SDS-PAGE and immunoblotting
The proteins were separated using SDS/PAGE (TGX gel, Bio-Rad) and transferred onto a PVDF membrane (Bio-Rad). The membrane was blocked in 5 % BSA and incubated for 18 h with the primary antibodies and subsequently with the respective secondary antibodies. Thereafter, the blot was developed using the ECL reagent (GE Healthcare) and immunoreactive signals were detected using the LAS-3000 imaging system (FUJIFILM Wako).

Preparation of total RNA and cDNA
Tissue samples were dissected and immediately frozen on dry ice. The tissue or cell samples were homogenised in QIAzol Lysis Reagent (QIAGEN), and total RNA was isolated using RNeasy kit purification (QIAGEN). First-strand cDNA synthesised was performed using the ReverTra Ace® qPCR RT Master Mix (Toyobo).

Measurement of mRNA levels
To determine the relative mRNA expression levels, a quantitative polymerase chain reaction was performed using the TaqMan gene expression assay (Nphs2, Rn00709834_m1; Cyp27b1, Rn01647147_g1; Rn28s as an internal control, Rn03034784_g1). The expression level relative to that in the vehicle-treated group was calculated using the comparative CT (2 -ΔΔCT ) method (User Bulletin #2, Applied Biosystems). For the comparative CT method, the ΔCT value was determined by subtracting the average of the CT value for the internal control from the average of the CT value for the target. The absolute copy number of the target gene was determined using a standard curve generated by amplifying known concentrations of synthetic oligonucleotides. The oligonucleotide primers, dual-labelled (FAM-TAMRA) oligonucleotide probes, and standard oligonucleotides were synthesised by Sigma-Aldrich (Supplementary Table 8 and  Supplementary Table 9). Amplification was performed on an ABI PRISM 7900HT Sequence Detector (Thermo Fisher Scientific) using the EXPRESS qPCR Supermix (Thermo Fisher Scientific) or THUNDERBIRD® qPCR Mix (Toyobo) as recommended by the manufacturers.