An animal model of type A cystinuria due to spontaneous mutation in 129S2/SvPasCrl mice.

Cystinuria is an autosomal recessive disease caused by the mutation of either SLC3A1 gene encoding for rBAT (type A cystinuria) or SLC7A9 gene encoding for b0,+AT (type B cystinuria). Here, we evidenced in a commonly used congenic 129S2/SvPasCrl mouse substrain a dramatically high frequency of kidney stones that were similar to those of patients with cystinuria. Most of 129S2/SvPasCrl exhibited pathognomonic cystine crystals in urine and an aminoaciduria profile similar to that of patients with cystinuria. In addition, we observed a heterogeneous inflammatory infiltrate and cystine tubular casts in the kidney of cystinuric mice. As compared to another classical mouse strain, C57BL/6J mice, 129S2/SvPasCrl mice had an increased mortality associated with bilateral obstructive hydronephrosis. In 129S2/SvPasCrl mice, the heavy subunit rBAT of the tetrameric transporter of dibasic amino acids was absent in proximal tubules and we identified a single pathogenic mutation in a highly conserved region of the Slc3a1 gene. This novel mouse model mimicking human disease would allow us further pathophysiological studies and may be useful to analyse the crystal/tissue interactions in cystinuria

LC–MS/MS-based quantification of efflux transporter proteins at the BBB

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Targeted unlabeled multiple reaction monitoring analysis of cell markers for the study of sample heterogeneity in isolated rat brain cortical microvessels

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Survival study and kidney injury in 129S2/SvPasCrl and C57BL/6J mice.

<p><b>A</b>, Survival proportions of mice from both strains before sacrifice at 16 weeks. 129S2/SvPasCrl mortality was significantly increased (p = 0.045) and necropsy revealed kidney dilation due to urolithiasis. <b>B</b>, Infrared cartography of 129S2/SvPasCrl kidney slices revealed the presence of focal cystine aggregates in renal tissues (cystine tubular casts). <b>C</b>, Renal function as assessed by enzymatic serum creatinine dosage was not significantly impaired in 129S1/SvPasCrl mice (n = 6) compared to C57BL/6J mice (n = 9) at 16 weeks (p = 0.11). <b>D–F</b>, Macrophage infiltrate in kidney tissues was assessed by morphometric analyses of F4-80 immunostaining and was increased in 129S2/SvPasCrl mice in comparison with C57BL/6J (n = 5/group, p = 0.046). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102700#pone-0102700-g003" target="_blank">Figures 3E</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102700#pone-0102700-g003" target="_blank">3F</a> are representative of macrophage infiltrates in C57BL/6 and 129S1/SvPasCrl mice respectively (magnification x200+ zoom). <b>G</b>, Fibrosis assessed by sirius red morphometric analysis did not evidence significant fibrosis amount in both strains (percentage of fibrotic area, p = NS).</p

129S2/SvPasCrl crystalluria and aminoaciduria are similar to those of human patients with cystinuria.

<p><b>A–B</b>, Typical flat hexagonal cystine crystals were present in 129S2/SvPasCrl fresh urine. <b>C</b>, Ninety percent of 129S2/SvPasCrl mice presented crystalluria (p = 0.0001 vs C57BL/6J mice). <b>D</b>, Urinary aminoacid chromatography has been performed in 129S2/SvPasCrl mice (n = 5) and C57BL/6J mice (n = 3). Dibasic aminoacids including cysteine (reduced form of cystine) were significantly higher in 129S2/SvPasCrl mice urine (p = 0.036 for each dibasic aminoacid) whereas other aminoacid renal excretion was similar in both strains. Some representative aminoacids are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102700#pone-0102700-g001" target="_blank">Figure 1D</a>. Results are expressed as aminoacid concentration (µMol)/creatinine concentration (mMol) in urines.</p

129S2/SvPasCrl mouse is affected by cystine urolithiasis.

<p><b>A</b>, Eighty percent of 129S2/SvPasCrl mice sacrificed at 16 weeks or dead before 16 weeks have been affected by urolithiasis whereas no C57BL/6J mice has been affected, p = 0.0007, n = 10/group. <b>B</b>, Bladder stones in a 129S2/SvPasCrl mouse. <b>C</b>, Spectrum of cystine was obtained by FTIR analysis of mouse stones. <b>D–F</b>, Scanning electron microscopy of a stone, at x58, x506, and x1070 magnification respectively, revealed the typical flat hexagonal structure of cystine crystals.</p

mRNA and protein expression of cystine transporters <i>Slc3a1</i>/rBAT and <i>Slc7a9</i>/b^0,+AT in kidney cortex from 129S2/SvPasCrl and C57bBL/6J mice.

<p><b>A–B</b>, Quantitative PCR: <i>Slc3a1</i> and <i>Slc7a9</i> mRNA expression was similar in both strains. <b>C</b>, Western Blot: b^0,+AT was expressed at a similar level in kidney cortex from both strains. <b>D</b>, Western Blot: rBAT was expressed in C57BL/6 mice but not in 129S2/SvPasCrl mice. <b>E–H</b>, Antibodies directed against the extracellular part of rBAT (Figures 4E and 4G) or against its intracellular part (Figures 4F and 4H) revealed the presence of rBAT at the brush border of proximal tubular cells in C57BL/6J mice (4E and 4F) but not in 129S2/SvPasCrl mice (4G and 4H).</p