Renal toxicity and biokinetics models after repeated uranium instillation

Abstract

During nuclear fuel processing, workers can potentially be exposed to repeated inhalations of uranium compounds. Uranium nephrotoxicity is well documented after acute uranium intake, but it is controversial after long-term or protracted exposure. This study aims to analyze the nephrotoxicity threshold after repeated uranium exposure through upper airways and to investigate the resulting uranium biokinetics in comparison to reference models. Mice (C57BL/6J) were exposed to uranyl nitrate (0.03-3 mg/kg/day) via intranasal instillation four times a week for two weeks. Concentrations of uranium in urines and tissues were measured at regular time points (from day 1 to 91 post-exposure). At each exposure level, the amount of uranium retained in organs/tissues (kidney, lung, bone, nasal compartment, carcass) and excreta (urine, feces) reflected the two consecutive weeks of instillation except for renal uranium retention for the highest uranium dose. Nephrotoxicity biomarkers, KIM-1, clusterin and osteopontin, are induced from day 4 to day 21 and associated with changes in renal function (arterial fluxes) measured using non-invasive functional imaging (Doppler-ultrasonography) and confirmed by renal histopathological analysis. These results suggest that specific biokinetic models should be developed to consider altered uranium excretion and retention in kidney due to nephrotoxicity. The threshold is between 0.25 and 1 mg/kg/day after repeated exposure to uranium via upper airways.

Figure 1

Uranium content in lungs, bones, kidneys, urine, feces and remaining carcass after repeated intranasal instillation (0.25–3 mg/kg/day) from Day 2 to Day 91 after the first instillation. Uranium concentration was evaluated in the tissue by ICP-MS and expressed in ng/g tissue wet weight. The values are expressed as mean ± SD. NE non-exposed. *p < 0.05/**p < 0.01/***p < 0.001, comparison with unexposed animals, Holm-Sidak test.

Figure 2

Uranium tissue content of mice exposed to intranasal instillation. (a) Uranium retention in lungs, kidneys, gastrointestinal tract, and nasal compartments 1 h after the first instillation to uranium of 0.25 mg/kg. Each point represents an independent animal, and the square represents the SAAM II (Simulation, analysis, and modeling software for tracer and pharmacokinetic studies) biodosimetric model, n = 4. GIT gastro-intestinal tractus, NC nasal compartment. (b) Biodosimetric model of uranium content in the kidneys after repeated intranasal instillations (0.03–3 mg/kg/day) according to the quantity of uranium administered to mice. Each point represents an independent animal; n = 4–12 for each time and dose.

Figure 3

Renal morphological and functional follow-up by ultrasound. A-C: Representative ultrasound images of the left kidney in mice exposed to uranium at 3 mg/kg/day for 91 days. B-mode (a), Color-mode (b) and corresponding Pulse-Wave doppler of the intrarenal artery (c) on Vevo-Lab® software (V5.6.1). D-E: Resistive index (RI) (d) and Pulsatile index (PI) (e) levels of intrarenal arteries obtained for mice before exposure (green), during exposure (D4), just after exposure (D11) and 3 months after exposure (D91) at 0.25 or 3 mg/kg/day. Each point represents an independent animal. The pre-instillation group (D0) includes all animals that were monitored for 4, 11 or 91 days after the first instillation to uranium or the vehicle solution. The non-exposed group (NE) received the vehicle solution (sodium bicarbonate) at the same time point as uranium exposed animals. n = 8 for each time and dose. The values are expressed as mean ± SEM. *P < 0.05/**P < 0.01/***P < 0.001, comparison with unexposed animals, Holm-Sidak test.

Figure 4

Assessment of renal damage by histopathological examination after exposure to uranium (0.25–3 mg/kg/day) by intranasal instillation. (a–e) Kidney longitudinal section microphotographs (200×) representative of damage observed after HES staining, scale bar = 100 µm. (a) Normal kidney. (b) Glomerulosclerosis and interstitial fibrosis. (c) Interstitial inflammation. (d) Tubular regeneration. (e) Tubular necrosis. (f) Total damage score (all criteria combined) according to exposure dose and time since first instillation. Each point represents an independent animal. (g,h) Percentage distribution of the different impairments for animals exposed to 1 mg/kg/day (g) or 3 mg/kg/day (h). The values are expressed as mean ± SD, n = 3–4 for each time and dose. *P < 0.05, comparison with unexposed animals, Two-way ANOVA.

Figure 5

Urine assay and immunostaining for KIM-1 after 0.25–3 mg/kg/day uranium exposure. Urine is collected by passing through a metabolic cage 16 h before the euthanasia of the animals. The kidneys are collected after the euthanasia. KIM-1 expression is measured in urine using ELISA (a) and by immunohistochemistry on a longitudinal kidney section (b–f). (b) Mean score over 10 fields according to exposure dose and time since first instillation. (e,f) Representative microphotographs corresponding to each score from 0 to 4, scale bar = 100 µm. NE non-exposed group. The values are expressed as mean ± SD, n = 3–4 for each time and dose. *P < 0.05/**P < 0.01/***P < 0.001, comparison with unexposed animals, Two-way ANOVA.

Figure 6

Gene expression of nephrotoxicity biomarkers in the renal tissue after uranium exposure (0.25–3 mg/kg/day). Results are expressed as a ratio to the expression of the housekeeping gene HPRT. AU arbitrary unit, B2M β-2 microglobulin, CLU clusterin, CST cystatin, KIM-1 kidney injury molecule 1, KLK kallikrein, NGAL lipocalin 2, OPN osteopontin. The values are expressed as mean ± SD, n = 3–4 for each time and dose. *P < 0.05/**P < 0.01/***P < 0.001, comparison to unexposed animals, Two-way ANOVA.