Uric acid suppresses 1 alpha hydroxylase in vitro and in vivo

Abstract

Objective: Patients with gout have lower calcitriol levels that improve when uric acid is lowered. The mechanism of these observations is unknown. We hypothesized that uric acid inhibits 1-αhydroxylase.

Materials and methods: In vivo, Sprague Dawley rats were randomized to control (n = 5), allantoxanamide (n=8), febuxostat (n=5), or allantoxanamide+febuxostat (n = 7). Vitamin D, PTH, and 1-αhydroxylase protein were evaluated. In order to directly evaluate the effect of uric acid on 1-αhydroxylase, we conducted a series of dose response and time course experiments in vitro. Nuclear factor κ-B (NFκB) was inhibited pharmacologically. Finally, to evaluate the potential implications of these findings in humans, the association between uric acid and PTH in humans was evaluated in a cross-sectional analysis of data from the NHANES (2003-2006); n = 9773.

Results: 1,25(OH)2D and 1-αhydroxylase protein were reduced in hyperuricemic rats and improved with febuxostat treatment. Uric acid suppressed 1-αhydroxylase protein and mRNA expression in proximal tubular cells. This was prevented by NFκB inhibition. In humans, for every 1mg/dL increase in uric acid, the adjusted odds ratio for an elevated PTH (>65 pg/mL) was 1.21 (95% C.I. 1.14, 1.28; P<0.0001), 1.15 (95% C.I. 1.08, 1.22; P<0.0001), and 1.16 (95% C.I. 1.03, 1.31; P = 0.02) for all subjects, subjects with estimated GFR ≥ 60, and subjects with estimated GFR <60 mL/min/1.73 m(2) respectively.

Conclusion: Hyperuricemia suppresses 1-αhydroxylase leading to lower 1,25(OH)2D and higher PTH in rats. Our results suggest this is mediated by NFκB. The association between uric acid and PTH in NHANES suggests potential implications for human disease.

Keywords: 1,25 dihydroxy-vitamin D; 1,25(OH)(2)D; 25 hydroxyvitamin D; 25(OH)D; ATX; BMI; CKD; FBXT; GFR; IQR; MBD; MDRD; Mineral and bone disorder; Mineral and bone disorders; Modification of Diet in Renal Disease; NFκB; NHANES; National Health and Nutrition Examination Survey; PTH; Parathyroid hormone; Uric acid; allantoxanamide; body mass index; chronic kidney disease; febuxostat; glomerular filtration rate; inter-quartile range; nuclear factor κ- B; parathyroid hormone.

Figure 1

Allantoxanamide successfully induces acute hyperuricemia and significantly inhibits 1-α hydroxylase activity in rats. Figure 1A: After 24 hours, mean serum uric acid was 1.9 ± 0.1, 3.3 ± 1.4, 1.3 ± 0.2, and 1.2 ± 0.3 mg/dL for the control, allantoxanamide, febuxostat, and allantoxanamide + febuxostat groups respectively. Figure 1B: Acute hyperuricemia leads to reduced 1-α hydroxylase activity estimated by the ratio of 1,25(OH)₂D to 25(OH)D. Figure 1C: Mean ± SD of 1,25(OH)₂D levels (pmol/L) in different treatment groups. Figure 1D: Mean ± SD of 25(OH)D levels (nmol/L) according to treatment group. CTRL: control, ATX: allantoxanamide, FBXT: febuxostat, and ATX + FBXT: allantoxanamide + febuxostat.

Figure 2

1-α hydroxylase protein expression is significantly reduced in hyperuricemic rats. Acute hyperuricemia had no significant effect on 24-hydroxylase protein expression. Figure 2A: Immunofluorescence for 1- α hydroxylase protein according to treatment group. Figure 2B: Western blotting for whole kidney lysates showing the expression of 1-α hydroxylase, 24- hydroxylase, and GAPDH proteins. The results shown are representative of all the animals included in the study. Figure 2C: Densitometry results comparing 1- α hydroxylase protein expression after normalization to GAPDH protein expression between the different treatment groups. The densitometry summarizes normalized 1-α hydroxylase protein expression for all the animals included in the study. CTRL: control, ATX: allantoxanamide, FBXT: febuxostat, and ATX + FBXT: allantoxanamide. + febuxostat.

Figure 3

Allantoxanamide treatment was associated with increased serum creatinine and tubular dilatation. Serum creatinine for the hyperuricemic group was significantly higher than the control group (1.9±1.2 versus 0.4±0.1 mg/dL). The acute kidney injury was partially reversed by the xanthine oxidase inhibitor, febuxostat (creatinine 0.7±0.2 mg/dL). Figure 3A: Mean ± SD of serum creatinine (mg/dL) are shown in the different treatment groups, Figure 3B: Allantoxanamide was associated with acute tubular toxicity. CTRL: control, ATX: allantoxanamide, FBXT: febuxostat, and ATX + FBXT: allantoxanamide. + febuxostat.

Figure 4

Uric acid suppresses 1-α hydroxylase mRNA and protein expression in human proximal tubular and inhibition of NFκB ameliorates the suppressive effect of uric acid on 1-α hydroxylase mRNA and protein. Figure 4A: 1- α hydroxylase protein expression is increasingly suppressed with increasing treatment dose of uric acid. Figure 4B: 1- α hydroxylase mRNA expression is suppressed with increasing treatment dose of uric acid. Figure 4C: 1-α hydroxylase protein expression is evident after 16 hours of treatment with uric acid (10mg/dL). Figure 4D: 1-α hydroxylase mRNA expression is significantly reduced after 6 hours of uric acid treatment (10mg/dL). Figure 4E: Inhibition of NFκB prevents the suppressive effect of uric acid on 1-α hydroxylase protein; Bay 11-7082 dose= 5 uM/mL, uric acid dose=10 mg/dL. Figure 4F: Inhibition of NFκB prevents the suppressive effect of uric acid on 1-α hydroxylase transcription. Results for each figure represent the findings of 3 independent experiments.