Urinary Copper Elevation in a Mouse Model of Wilson's Disease Is a Regulated Process to Specifically Decrease the Hepatic Copper Load

Abstract

Body copper homeostasis is regulated by the liver, which removes excess copper via bile. In Wilson's disease (WD), this function is disrupted due to inactivation of the copper transporter ATP7B resulting in hepatic copper overload. High urinary copper is a diagnostic feature of WD linked to liver malfunction; the mechanism behind urinary copper elevation is not fully understood. Using Positron Emission Tomography-Computed Tomography (PET-CT) imaging of live Atp7b−/− mice at different stages of disease, a longitudinal metal analysis, and characterization of copper-binding molecules, we show that urinary copper elevation is a specific regulatory process mediated by distinct molecules. PET-CT and atomic absorption spectroscopy directly demonstrate an age-dependent decrease in the capacity of Atp7b−/− livers to accumulate copper, concomitant with an increase in urinary copper. This reciprocal relationship is specific for copper, indicating that cell necrosis is not the primary cause for the initial phase of metal elevation in the urine. Instead, the urinary copper increase is associated with the down-regulation of the copper-transporter Ctr1 in the liver and appearance of a 2 kDa Small Copper Carrier, SCC, in the urine. SCC is also elevated in the urine of the liver-specific Ctr1 −/− knockouts, which have normal ATP7B function, suggesting that SCC is a normal metabolite carrying copper in the serum. In agreement with this hypothesis, partially purified SCC-Cu competes with free copper for uptake by Ctr1. Thus, hepatic down-regulation of Ctr1 allows switching to an SCC-mediated removal of copper via kidney when liver function is impaired. These results demonstrate that the body regulates copper export through more than one mechanism; better understanding of urinary copper excretion may contribute to an improved diagnosis and monitoring of WD.

Age-dependent changes of cerebral copper metabolism in Atp7b −/− knockout mouse model of Wilson’s disease by [64Cu]CuCl2-PET/CT

“…[ 64 Cu]CuCl 2 -PET/CT imaging of Balb/c mice was performed using a method described previously [7–9, 13]. Briefly, Balb/c mice were anesthetized by 3% isoflurane in 100% oxygen (3 L/min) at room temperature, using an isoflurane vaporizer (Summit Anesthesia Solutions, Salt Lake City, UT).…”

“…Positron emission tomography (PET) is a useful technology for functional imaging of cerebral metabolic activity in vivo based on its high sensitivity and quantification capability. Recently, biodistribution and radiation dosimetry of [ 64 Cu]CuCl 2 in Atp7b −/− knockout mice, a well-established mouse model of WD [6], was determined by PET/CT analysis [7–9]. Exploring feasibility and use of [ 64 Cu]CuCl 2 as a tracer for noninvasive assessment of changes of cerebral copper metabolism in WD, this study aimed to assess age-dependent changes of 64 Cu radioactivity in the brains of Atp7b −/− knockout mice using PET/CT, following intravenous injection of [ 64 Cu]CuCl 2 as a tracer.…”

“…In order to determine whether [ 64 Cu]CuCl 2 can be used as a tracer for assessment of cerebral copper metabolism, a sequential PET/CT imaging was conducted to compare biodistribution and cerebral [ 18 F]FDG and 64 Cu radioactivity in mice intravenously injected with [ 18 F]FDG and [ 64 Cu]CuCl 2 as a tracer, respectively. Continuing from recent studies of 64 Cu biodistribution and radiation dosimetry in the Atp7b −/− knockout mice administered with [ 64 Cu]CuCl 2 intravenously [7] or orally [8, 9], further PET quantitative analysis was performed to measure age-dependent changes of 64 Cu radioactivity in the brains of Atp7b −/− knockout mice. The data from this preclinical study support further development of [ 64 Cu]CuCl 2 as a new tracer for assessment of age-dependent changes of copper metabolism in WD patients presented with neuropsychiatric symptoms using a PET or PET/CT scanner.…”

Copper trafficking to the secretory pathway

“…When added to HEK293 cells, SCC competed with the radioactive Cu for the uptake by Ctr1, further indicating that SCC could be a source of Cu for cells. 22 Finally, experiments with cultured cells first loaded with Cu and then washed and placed in a serum-free medium revealed that cells secrete Cu in a complex with SCC or the SCC-like molecule. This result is interesting, as it suggests that SCC interacts and forms complex with Cu within the cell, probably within the secretory pathway.…”

Zinc in denture adhesive: a rare cause of copper deficiency in a patient on home parenteral nutrition

“…Approximately 50% of copper is excreted in bile and the rest in other intestinal secretions (saliva, gastric, pancreatic and small intestinal). Normally, 10–15% of copper in bile is reabsorbed, but this is an important mechanism of copper homoeostasis and biliary excretion declines in hypocupraemia,7 and, conversely, biliary excretion increases in the presence of high free copper levels in blood, through the action of ATP7B, the copper transporter inactivated in Wilson's disease 8. If this second hypothesis is correct, it shows the importance of the secretion of free copper into the gut, even when given intravenously in large amounts.…”