Fig 1: Growth retardation and renal phenotype in a whole body OcrlY/- mouse model. (A) Dot plot representing the measurement of body weight, (B) albumin and (D) CC16 urinary excretion in Ocrl male mice at 8, 16, 24 and 48 weeks after birth (8 weeks, n = 7 mice per group; 16 weeks, n = 7 mice per group; 24 weeks, n = 7 mice per group; 48 weeks, n = 5 mice per group). (C) Coomassie blue-stained SDS-PAGE analysis of urine derived from 8-week-old Ocrl mice and densitometry quantification of albumin (n = 3 mice per group). A total of 10 µg of BSA were loaded as positive control [first lane; molecular weight, ~66.5 kDa; arrowhead]. (E) Representative western blotting and densitometry quantification of TFR, VDBP and CC16 in urine derived from 8-week-old Ocrl mice (TFR, VDBP and CC16, n = 5 mice per group). (F) Quantification of CC16 urinary excretion in 8-weeks-old Clcn5 and Ocrl mice (n = 5 mice per group). All the urine parameters were normalized to urinary creatinine concentration. Plotted data represent mean ± SEM. Each dot of the graphs represents one mouse. Two-tailed unpaired Student’s t-test, *P < 0.05, **P < 0.01 and ***P < 0.001 relative to OcrlY/+ or Clcn5Y/+ mice. ns: not significant.
Fig 2: Temporal mTORC1 activation by cystine buildup leads to kidney disease development in CTNS-deficient mice.a CC16 levels in the urine of Ctns mice at the indicated times; 6 weeks: n = 7 CtnsWT and n = 8 CtnsKO animals; 12 weeks: n = 11 CtnsWT and n = 9 CtnsKO animals; 16 weeks: n = 8 CtnsWT and n = 10 CtnsKO animals; 24 weeks: n = 8 CtnsWT and n = 10 CtnsKO animals; 32 weeks: n = 9 CtnsWT and n = 7 CtnsKO animals. b Cystine levels in Ctns mouse kidneys; 6 weeks: n = 9 animals in each experiment group; 12 weeks: n = 8 CtnsWT and n = 6 CtnsKO animals; 24 weeks: n = 5 CtnsWT and n = 4 CtnsKO animals. c Schematic of the experimental workflow for monitoring mTORC1 activity in Ctns mice and in cultured PT cells. mPTCs from (d, f) asymptomatic (6 weeks of age), (e, g) pre-symptomatic (12 weeks of age), and i symptomatic (24 weeks of age) CtnsKO mice and their (age-matched) CtnsWT littermates were cultured under fed and starved conditions. (d, e) Immunoblotting and quantification of the indicated proteins; n = 3 biologically independent animals. (f, g) Confocal microscopy and quantification of mTOR+-Lamp1+ signals; n = 10 randomly selected and non-overlapping fields of views from 3 biologically independent animals. Nuclei counterstained with DAPI (blue). h Fed mPTCs were treated with cysteamine (100 nM) or Torin1 (250 nM) for 16 h. Cysteamine enters the lysosomes through a distinct importer and reacts with cystine to exchange its disulfide bridge into a mixed (cysteamine-cysteine) disulfide, which exits via the cationic amino-acid exporter PQLC2, and generates free cysteine, which exits from lysosomes through an unknown transporter. i Immunoblotting of the indicated proteins; n = 2 biologically independent experiments. j Cystine levels in Ctns mPTCs; n = 6 biologically independent samples. Plots represent mean ± SEM. Statistics were calculated by unpaired two-tailed Student’s t test. Scale bars, 10 µm. Source data are provided as a Source Data file. Images in c, h were drawn using pictures from Servier Medical Art, which is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).
Fig 3: Cystine storage diverts differentiation towards growth and proliferation.a Cystine levels in Ctns mouse kidneys; n = 6 animals in each experimental group. b Immunofluorescence and quantification of the Lamp1 mean fluorescence intensity (MFI) in the LTL+ PT segments of Ctns mouse kidneys, n = 164 CtnsWT and n = 137 CtnsKO PT segments from 3 biologically independent animals. c Confocal microscopy and quantification of the Cy5-tagged ß-lactoglobulin MFI in the LTL+ PT segments of Ctns mouse kidneys, n = 356 CtnsWT and n = 330 CtnsKO PT segments from 3 biologically independent animals. d Quantification of LMW/Clara cell protein (CC16) levels in the urine samples of Ctns mice; n = 7 animals in each experimental group. e Volcano plot of genome-wide changes in PT segments of Ctns mouse kidneys. Red and green dots show the differentially expressed genes (DEGs; adjusted p value < 0.05; n = 3 animals in each experimental group). f GSEA of over- and under-represented biological pathways in the PT segments of CtnsKO mouse kidneys. FDR q value was calculated from the enrichment pathway analysis in NetworkAnalyst. g Immunoblotting and quantification of the indicated proteins in the kidneys of Ctns mice; n = 3 animals in each experimental group. h Confocal microscopy of whole-kidney sections stained for Ccna2 (red), Lrp2 (blue), and PT marker (LTL, green); quantification of Ccna2+ nuclei and of Lrp2 MFI in the PT segments of Ctns mouse kidneys. For Ccna2+ nuclei: n = 31 CtnsWT and n = 54 CtnsKO PT segments from 3 biologically independent animals. For Lrp2 MFI: n = 60 CtnsWT and n = 56 CtnsKO PT segments from 3 biologically independent animals. i mRNA levels for the indicated genes in the kidneys of Ctns mice; n = 8 biologically independent animals. j Whole-kidney sections stained for PT apical marker (Aqp1, green) and PT basolateral marker (Laminin, red). Confocal microscopy and quantification of PT cell height in the kidneys of Ctns mouse; n = 90 PT segments from 3 biologically independent animals. Plots represent mean ± SEM. Statistics calculated by unpaired two-tailed Student’s t test. Dotted white squares contain images at high magnification. Nuclei counterstained with DAPI (grey). Scale bars are 50 µm in b, c, and h and 20 µm in j. Source data are provided as a Source Data file.
Supplier Page from Biomatik for Mouse Clara Cell Protein 16 (CC16) ELISA Kit