Renal function is altered in <i>Atp7b</i>^−/− mice older than 20 weeks.

<p>Water intake (A), Food intake (B) and urine volume output (C) relative to age-matched wild-type mice were measured over a 24 hr period. Double asterisks denote p≤0.003. N = 3 to 7 age-matched pairs; data presented as mean ± SD; additional supporting data and details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038327#pone.0038327.s001" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038327#pone.0038327.s005" target="_blank">information S1</a>.</p

<i>Atp7b</i> inactivation induces age-dependent changes in urine copper content.

<p>(A) Urinary copper concentration and (B) total amount from wild-type (WT) and <i>Atp7b</i>^−/− (KO) mice at different ages. Urine collected over 24 hours and measured by atomic absorption. Data reported as mean concentration ± SD, n = 3 to 13, animals per age group. *P<0.05 versus age-matched wild type. Boxplot middle horizontal bar represents the median; the dotted horizontal line signifies the mean. The while upper and lower box values signify the 75^th and 25^th percentiles, whiskers represent the 10^th and 90^th percentiles. The black dots represent outliers (three SD from the mean).</p

PET-CT analysis of ⁶⁴Cu distribution in live mice at 24 hrs after oral administration of ⁶⁴CuCl₂.

<p>Representative PET-CT images of (A) <i>Atp7b</i>^−/− and (B) wild-type mice at 24 hours post oral administration (PO) of ⁶⁴CuCl₂. Orange-brown color denotes ⁶⁴Cu radioactivity. Red arrows and white arrows identify liver and gastrointestinal tract ⁶⁴Cu radioactivity, respectively. (C) Hepatic ⁶⁴Cu radioactivity in <i>Atp7b^−/−</i> and wild-type mice at 24 hrs PO (p<0.001). Age-dependent ⁶⁴Cu radioactivity in the (D) kidneys (p = 0.38 for group effect) and (E) urinary bladder of wild-type and <i>Atp7b</i>^−/− mice at 24 hr PO. %ID/g, percentage of administration dose per gram. Data presented as mean ± SD. N = 5, number of mice with same age and genotype. Additional data and details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038327#pone.0038327.s002" target="_blank">Figure S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038327#pone.0038327.s005" target="_blank">Information S1</a>.</p

Characterization of urine copper component(s).

<p>(A) Representative graph of gel filtration analysis on 20 µl of <i>Atp7b</i>^−/− urine along with copper levels in resulting fractions. (B) % of total copper found in the filtrate or retentate of wild-type and <i>Atp7b</i>^−/− urine after application of whole urine to a filter with 3 kDa cutoff. Ages varied from 6 to 65 weeks. Data presented as % of total copper ± SD, n = 21 age-matched wild-type/<i>Atp7b</i>^−/− pairs. (C) Representative graph of copper profile from gel filtration of 3 kDa filtrate from wild-type and <i>Atp7b</i>^−/− urine. Arrows point to the elution time of indicated compounds.</p

Elemental content of wild-type and <i>Atp7b</i>^−/− urine during disease progression.

<p>ICP-MS analysis of urine collected over 24 hours from age-matched wild-type and <i>Atp7b</i>^−/− mice. (A) Comparison of WT and KO copper amounts at different ages. (B) Urine amounts of Na, Mg, P, K, Ca, Se, and urine volume (relative to wild-type) at different ages. (C) Amounts of Fe and Zn at different ages (relative to wild type). N = 2 to 4 age-match WT/KO pairs for each time point. Data presented as mean ± SD, **P≤0.003. The data points (from left to right) in Panel A represent 6 weeks, 12 weeks, 15 to 20 weeks, and 60 to 65 weeks. Panels B and C data points (from left to right) represent 6 to 12 weeks, 15 to 20 weeks, and 60 to 65 weeks. Correlation coefficients found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038327#pone.0038327.s004" target="_blank">Table S1</a>.</p

Ctr1 and Atp7A levels in the liver and kidney of Atp7b^−/− and wild-type mice.

<p>(A) Real-time PCR analysis of Atp7A mRNA levels in the liver of wild-type and Atp7b^−/− mice at different ages. Data presented as mean ± SD, n = 3 to 4 different samples of each genotype and age. (B) Representative Western blot illustrating ATP7A protein expression in the livers of wild-type and <i>Atp7b</i>^−/− mice at 7 and 20 weeks. Twenty weeks old <i>Atp7b</i>^−/− kidneys were used as a positive control for ATP7A protein expression. “ST” denotes molecular weight standards. Black arrow points to ATP7A band. Weak bands in the liver samples are due to non-specific staining (C, D) Quantitation of CTR1 protein levels by Western blot analysis and densitometry (relative to wild type) in liver and kidneys of wild-type and <i>Atp7b</i>^−/− mice at different ages. Band intensity in each sample was normalized to a β-actin loading control. Data presented as mean ± SD. n = 2 for each genotype per age group.</p

Functional interaction of SCC and CTR1.

<p>(A) Copper concentrations in fractions obtained from gel filtration of wild-type, <i>Atp7b</i>^−/−, and liver-specific <i>Ctr1</i>^−/− urine. (B) 64Cu uptake assay by HEK293 cells stably overexpressing hCtr1 in the presence of increasing concentrations of gel filtration purified SCC from urine of <i>Atp7b</i>^−/− and wild-type mice. Radioactive copper-64 was kept constant at 0.25 µM. Sodium phosphate buffer pH 7.4 was used as the buffer control. 1× is equal to a copper concentration of 0.25 µM for <i>Atp7b^−/−</i> SCC and 2× to 20× are multiples of the original 1× concentration. The volumes used for the assays were set to achieve the desired 1× to 20× concentrations specifically for <i>Atp7b^−/−</i> SCC. The wild-type SCC 1× to 20× assays are done with the same volumes of SCC utilized for corresponding <i>Atp7b^−/−</i> SCC assays, thus wild-type concentrations used are low due to inherent lower copper levels in WT SCC. Data presented as mean ± SD, performed in triplicates. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038327#pone.0038327.s003" target="_blank">Figure S3</a>.</p