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

Abstract

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.

Fig. 1. SC-919 is an effective inhibitor of IP6K in vivo.

a IP6K-mediated synthesis of the inositol pyrophosphates. PIP₂ phosphatidylinositol 4,5-bisphosphate, Ins inositol; the subscripts denote the total number of phosphates; PPIP5K diphosphoinositol pentakisphosphate kinase. b The chemical structure of SC-919. InsP₇ levels in the liver (c), muscle (d), and kidney (e) after 2 h of SC-919 administration to rats. Values indicate mean ± S.D. (n = 6 biological replicates). ^†P < 0.05 and ^‡P < 0.05 vs. vehicle as determined using Williams’ test and Shirley–Williams test, respectively. Veh vehicle.

Fig. 2. IP6K inhibition reduces the export of phosphate via an XPR1-dependent mechanism involving the SPX domain.

Effect of treatment with SC-919 for 4 h on the a intracellular InsP₇ levels in 293 cells, b exported extracellular ³²P activity, and c intracellular ³²P activity in the phosphate export assay in 293 cells, d intracellular ³²P activity in the phosphate uptake assay in 293 cells, e, f Levels of InsP₇ in HAP1 wild-type and XPR1 KO cells, g levels of exported extracellular ³²P activity, and h intracellular ³²P activity in phosphate export assay in HAP1 wild-type and XPR1 KO cells, i intracellular ³²P activity in phosphate uptake assay in HAP1 wild-type and XPR1 KO cells, j, k Exported extracellular ³²P activity and intracellular ³²P activity in phosphate export assay in XPR1 KO cells introduced with either eGFP, XPR1, or inositol pyrophosphates-binding site-deleted XPR1 (XPR1ΔSPX) treated with either DMSO or SC-919 (1 μM). Values indicate mean ± S.D. (n = 5 biological replicates for a–f and 6 biological replicates for g–k). ^†P < 0.05 and ^‡P < 0.05 vs. vehicle as determined using Williams’ test and Shirley–Williams test, respectively. **P < 0.01 as determined using Student’s t-test, and ^#P < 0.05 as determined using the Aspin–Welch test. D, DMSO. CPM, counts per minute.

Fig. 3. IP6K inhibition decreases plasma phosphate levels in rats and monkeys and does not inhibit phosphate intake by the gut or phosphate reuptake by the kidney in rats.

a Plasma concentration of SC-919 and b phosphate levels in normal rats administered SC-919. (n = 3 biological replicates for pharmacokinetics and n = 5 biological replicates for the determination of plasma phosphate levels). c, d Concentration of SC-919 and e phosphate levels in the plasma of normal monkeys administered SC-919. (n = 6 biological replicates). f Effect of SC-919 on the cumulative ³²P activity in the faeces of rats that were administered NaH₂³²PO₄ solution. (n = 5 biological replicates). g Effect of SC-919 on the cumulative ³²P activity in the urine of rats that were administered NaH₂³²PO₄ solution. (n = 5 biological replicates). Values indicate mean ± S.D. ^†P < 0.05 and ^‡P < 0.05 vs. vehicle as determined using Williams’ test and Shirley–Williams test, respectively. ^¶¶P < 0.01 and ^§P < 0.05 vs. vehicle as determined using Dunnett’s test and Steel test, respectively.

Fig. 4. IP6K inhibition decreases the plasma phosphate levels in rats with hyperphosphataemia.

a Concentration of SC-919 and b plasma phosphate levels following the initial dose of SC-919 or sevelamer in adenine-treated rats. (n = 3 biological replicates for pharmacokinetics and n = 6 and 5 biological replicates for adenine-treated rats and normal rats for determination of phosphate levels in the plasma). c Concentration of SC-919 and d plasma phosphate levels following the seventh dose of SC-919 or sevelamer in adenine-treated rats. (n = 3 biological replicates for pharmacokinetics and n = 6 and 5 biological replicates for adenine-treated rats and normal rats for determination of phosphate levels in the plasma). e Concentration of SC-919 and f plasma phosphate levels in bilaterally nephrectomized rats that were orally administered with SC-919. Animals found dead during the study (1 rat in SC-919 1 mg/kg of e, 2 rats in nephrectomized-vehicle, and 1 rat in nephrectomized-SC-919 1 mg/kg of f. (n = 5–7 biological replicates for nephrectomzed rats, and n = 6 biological replicates for sham-operated rats for e and f). Values indicate mean ± S.D. ^†P < 0.05 and ^‡P < 0.05 vs. vehicle as determined using a Williams’ test and the Shirley–Williams test, respectively. Sev sevelamer.

Fig. 5. IP6K inhibition increases the intracellular levels of ATP in an XPR1-dependent manner.

a Time course of ATP levels in SC-919-treated 293 cells. b Intracellular levels of ATP in HAP1 wild-type and XPR1 KO cells. Effects of the treatment with SC-919 after 4 h on c intracellular levels of ATP in HAP1 wild-type and XPR1 KO cells, d intracellular levels of ATP in XPR1 KO cells introduced with either eGFP, XPR1, or inositol pyrophosphates-binding site-deleted XPR1 (XPR1ΔSPX) treated with either DMSO or SC-919 (1 μM), e ³²P activity in ATP in 293 cells pre-treated with ³²P-phosphate followed by SC-919 treatment, f ³²P activity in ATP in 293 cells pre-treated with non-labelled phosphate followed by SC-919 plus ³²P-phosphate treatment. Values indicate mean ± S.D. (n = 5 biological replicates for a–c, 5–6 biological replicates for e, and 6 biological replicates for d, f). ^†P < 0.05 and ^‡P < 0.05 vs. vehicle as determined using Williams’ test and Shirley–Williams test, respectively. **P < 0.01 as determined using Student’s t-test, and ^##P < 0.01 as determined using the Aspin–Welch test. CPM, counts per minute.

Fig. 6. Chronic IP6K inhibition alleviates CKD-induced hyperphosphataemia and related parameters in adenine-treated rats.

Evaluation of the chronic effect of SC-919 in adenine-treated rats. a Experimental protocol for a–r. b Daily food intake. c Body weight. d Plasma GDF15 levels. e Plasma FGF23 levels. f Plasma 1,25(OH)₂-D levels. g Plasma intact PTH levels. h Plasma phosphate levels. i Plasma calcium-phosphate products. j Plasma creatinine levels. k Aortic calcium content. l Representative aortic images. Levels of the m Nphs2 and n Cyp27b1 mRNAs in the kidney. o Fibrotic and p non-fibrotic area in kidney. q Kidney collagen contents. r Representative kidney images (bar = 250 μm, each lower right square shows the entire kidney image). Animals found dead during the study (1 rat in vehicle, 1 rat in SC-919 10 mg/kg). (n = 11–12 biological replicates for adenine-treated rats and n = 5–6 biological replicates for normal rats for a–r). s Kidney ATP levels (6 h after the drug dose) in adenine-rats treated with SC-919 for 8 days. (n = 6 biological replicates). Values indicate mean ± S.D. ^†P < 0.05 and ^‡P < 0.05 vs. vehicle as determined using Williams’ test and Shirley–Williams test, respectively. *P < 0.05 and **P < 0.01 vs. vehicle as determined using Student’s t-test and ^#P < 0.05 and ^##P < 0.01 vs. vehicle as determined using the Aspin–Welch test. Sev, sevelamer.

Fig. 7. Schematic representation of IP6K-mediated phosphate regulation and its therapeutic relevance.

In the normal condition, IP6K stimulates cellular phosphate export via SPX domain of XPR1, maintaining plasma phosphate levels in vivo. IP6K-inhibition-mediated reduction of InsP₇ inhibits phosphate export ability of XPR1 in cells, resulting in decrease in plasma phosphate levels in vivo. IP6K inhibition is therapeutically relevant and improves hyperphosphataemia and associated complications. IP6K inositol hexakisphosphate kinase.