Digenic Inheritance in Cystinuria Mouse Model

<div><p>Cystinuria is an aminoaciduria caused by mutations in the genes that encode the two subunits of the amino acid transport system b^0,+, responsible for the renal reabsorption of cystine and dibasic amino acids. The clinical symptoms of cystinuria relate to nephrolithiasis, due to the precipitation of cystine in urine. Mutations in <i>SLC3A1</i>, which codes for the heavy subunit rBAT, cause cystinuria type A, whereas mutations in <i>SLC7A9</i>, which encodes the light subunit b^0,+AT, cause cystinuria type B. By crossing <i>Slc3a1</i>^<i>-/-</i> with <i>Slc7a9</i>^-/- mice we generated a type AB cystinuria mouse model to test digenic inheritance of cystinuria. The 9 genotypes obtained have been analyzed at early (2- and 5-months) and late stage (8-months) of the disease. Monitoring the lithiasic phenotype by X-ray, urine amino acid content analysis and protein expression studies have shown that double heterozygous mice (<i>Slc7a9</i>^+/-<i>Slc3a1</i>^+/-) present lower expression of system b^0,+ and higher hyperexcretion of cystine than single heterozygotes (<i>Slc7a9</i>^+/-<i>Slc3a1</i>^+/+ and <i>Slc7a9</i>^+/+<i>Slc3a1</i>^+/-) and give rise to lithiasis in 4% of the mice, demonstrating that cystinuria has a digenic inheritance in this mouse model. Moreover in this study it has been demonstrated a genotype/phenotype correlation in type AB cystinuria mouse model providing new insights for further molecular and genetic studies of cystinuria patients.</p></div

Western blot of brush‐border kidney membranes.

<p>Protein analysis of rBAT and b^0,+AT in kidney brush‐border membranes of different genotypes. Fifty micrograms of protein were loaded in all lanes and run in non‐reducing conditions in 7% acrylamide SDS-page. Molecular mass standard (kDa) are indicated. <b>A.</b> Antibodies against rBAT and β-actin were used and are indicated on the left side. Arrows from right side indicate the heterodimer (HT) and, as well the oligomeric structure of 2 heterodimers (2xHT). Wild‐type (<i>Slc7a9</i>^+/+<i>Slc3a1</i>^+/+), single mutants (<i>Slc7a9</i>^+/+<i>Slc3a1</i>^-/- and <i>Slc7a9</i>^-/-<i>Slc3a1</i>^+/+), double mutant (<i>Slc7a9</i>^-/-<i>Slc3a1</i>^-/-) and double heterozygous (<i>Slc7a9</i>^+/-<i>Slc3a1</i>^+/-) are shown. <b>B and C.</b> Antibodies against b^0,+AT and β-actin were used and are indicated on the left side. Arrows from right side indicate the rBAT/b^0,+AT heterodimer (HT), the oligomeric structure of 2 heterodimers (2xHT) and a band corresponding to a complex, variable among experiments, formed by the oligomerization of b^0,+AT detached from rBAT artifact of brush-border extraction and/or SDS-page conditions (*). (B) Wild type, single mutants, double mutant and double heterozygous mice are represented. (C) Wild type, double and both single heterozygous (<i>Slc7a9</i>^+/-<i>Slc3a1</i>^+/+ and <i>Slc7a9</i>^+/+<i>Slc3a1</i>^+/-) samples are shown. <b>D</b>. Graph representing the densitometry analysis of b^0,+AT corrected by β-actin of double and single heterozygous, rBAT mutant and double mutant samples compared to wild type. The mean of 3–4 independent western blot analysis is represented.</p

Inheritance percentage of F2 generation.

<p>Data from 1152 offspring obtained from 10 different couples of F2 double heterozygous progenitors. Male/Female: Number of animals of each genotype. Observed: Percentage of newborn for each genotype. Expected: Percentage of segregation according to Mendel inheritance. No differences were found analyzing data with Chi-square test (p-value = 0,98 comparing real and expected frequency).</p><p>Inheritance percentage of F2 generation.</p

Urine amino acid content.

<p>Mean±S.E.M. of urine amino acids (nmols amino acid/24h∙g) of arginine (<b>A</b>), ornithine (<b>B</b>), lysine (<b>C</b>) and cystine (<b>D</b>) from 3 months‐old male mice (n≥7 animals for each genotype) are represented. Student’s t-test is represented as * p<0,05; **p<0,01 and ***p<0,001. Bars which represent double heterozygous (<i>Slc7a9</i>^<i>+/-</i><i>Slc3a1</i>^<i>+/-</i>) and double mutants (<i>Slc7a9</i>^‐-/-<i>Slc3a1</i>^-/-) are filled in gray and black, respectively.</p

Histopathological studies of renal sections.

<p>Hematoxylin/eosine staining. Analysis were done in wild‐type mice (row A), single mutants (row B) and double mutant (row C) mice. In B and C groups; only kidneys with calculi were analyzed. Column 1 shows glomerulus (G). <b>A1</b>: normal glomerulus; <b>B1</b>: glomerulus with partial collapse of capillary loops; <b>C1</b>: glomerulus with global matrix increase. Column 2 shows interstitial inflammation (arrow) and tubular atrophy. <b>A2</b>: normal tissue; <b>B2</b>: high level of interstitial inflammation; <b>C2</b>: interstitial inflammation and tubular atrophy. Column 3 shows renal tubules (t). <b>A3</b>: normal renal cortex; <b>B3</b>: tubular atrophy in renal cortex; <b>C3</b>: tubular atrophy with lumen dilatation.</p

Percentage of calculi formation and lithiasis prevalence.

<p>Table shows the results of tracking the calculi formation by X-ray. Number of mice that have developed calculi in bladder, kidney or in both organs (Bladder and kidney) at 2, 5 and 8 months of age (2m, 5m and 8m) are indicated (mice with calculi / mice studied). Percentages of mice with calculi (% Lithiasis) and with simultaneous calculi formation (% Lithiasis in bladder and both kidneys) are indicated.</p><p>Percentage of calculi formation and lithiasis prevalence.</p

Plasma T4 and T3 and cortex T3 concentrations in mice of different genotypes and different ages, as indicated.

<p><i>Wt</i>  =  wild type mice; M8 =  <i>Mct8KO</i>. L2 =  <i>Lat2KO</i>; M8L2 =  <i>Mct8Lat2KO</i>. Significance of differences was calculated by one way ANOVA, and the Tukey posthoc test. Only relevant significant comparisons are indicated. * P<0.05. ** P<0.01. *** P<0.001. P0, P5, and P21: postnatal days 0, 5, and 21, respectively.</p

Type 1 deiodinase (<i>Dio1</i>) expression (mean ± SE) in the liver.

<p><i>Wt</i> =  wild type mice; M8 =  <i>Mct8KO</i>; L2 =  <i>Lat2KO</i>; M8L2 =  <i>Mct8Lat2KO</i>. Measurements were by qPCR, and the data expressed relative to 18S RNA. Note the different scale for the P0 data with respect to P5 and P21. Significance of differences was calculated by one way ANOVA and the Tukey posthoc test. Only relevant significant comparisons are indicated. ** P<0.01. *** P<0.001. P5, P21: postnatal days 5 or 21.</p

Gene expression (mean ± SE) in the cerebral cortex and liver of mice of different genotypes and ages as indicated.

<p><i>Wt</i> =  wild type mice; M8  =  <i>Mct8KO</i>; L2 =  <i>Lat2KO</i>; M8L2 =  <i>Mct8Lat2KO</i>. Measurements were by qPCR, and the data expressed relative to 18S RNA. Significance of differences was calculated by one way ANOVA and the Tukey posthoc test. Only relevant significant comparisons are indicated. * P<0.05. ** P<0.01. *** P<0.001. <i>Hr</i>: Hairless mRNA. <i>Sema7a</i>: Semaphorin 7a mRNA. <i>Klf9</i>: Kruppel factor 9, or BTEB mRNA. P0, P5, P15, P21: postnatal days 0, 5, 15, or 21.</p

SDS-PAGE, Western blot analysis, SEC and SPA of 4F2hc-LAT2 purified in DDM, LMNG and CHS.

<p>(A) Coomassie Blue-stained 10% SDS/polyacrylamide gel (lane C) of 4F2hc-LAT2 after the two affinity chromatography steps. The complex runs as a prominent band at ∼125 kDa. Western blot analysis using anti-4F2hc (lane α4F2_ED) and anti-StrepTagII (lane αSTag) antibodies indicated the presence of intact heterodimers only, i.e., no 4F2hc or LAT2 from disrupted complexes. (B) SEC of purified 4F2hc-LAT2 indicated a prominent almost symmetrical elution peak at 13.7 ml. (C) Silver-stained 10% SDS/polyacrylamide gel of purified 4F2hc-LAT2 after gel filtration (lane ST; from peak fraction). Again, one single band is visible corresponding to the heterodimer. Integrity of the complex was further supported by Western blotting using anti-4F2hc (lane α4F2_ED) and anti-StrepTagII (lane αSTag) antibodies. (D) Radioligand-binding assay by SPA using purified 4F2hc-LAT2 and [³H]L-leucine. Bar 1: Binding of the radiolabelled substrate L-leucine to 4F2hc-LAT2, which is bound to scintillation beads, induces SPA signal. As expected, SPA signal was abolished by addition of 4 mM cold L-leucine (bar 2; competitive inhibition) or 100 mM imidazole (bar 3; detachment of the protein from the SPA beads). Bars represent mean ± SEM from triplicates. One representative of three similar independent experiments is shown.</p