Fig 1.
Workflow for tracking and proteomic analysis of Slc3a1-/- mouse cystinuric stones.
Shows representative images for the tracking of mouse stones using μCT, collecting and solubilizing for mass spectrometry analysis using both DDA and DIA.
Fig 2.
(A) μCT imaging and 3D modeling of in vivo cystine stone development in a representative Slc3a1-/- mouse. (B) μCT imaging and 3D modeling of urinary stone growth in an individual Slc3a1-/- mouse. Stone formation often initiates as sediment in the bladder (P62), and progresses through agglomeration (P70–76) to form urinary stones (P84); this progression can be seen here. (C) μCT imaging and 3D modeling of a single stone’s growth in mm3 over 142 days.
Fig 3.
Proteomic analysis of stone development in large and sand kidney stones.
(A) Venn diagram comparing the proteins identified in sand-sized stones (698 proteins) versus large stones (577 proteins). We identified 426 proteins in both fractions. (B) Heatmap of all proteins that were significantly changed in abundance in small, medium, and large versus sand stones (q-value < 0.05) by at least 1.5-fold (200 proteins total). Heatmap colors represent the log2 fold-change of each protein versus the median value in the fraction.
Fig 4.
Pathway analysis of significantly changed proteins comparing large stones vs sand.
(A) Among upregulated pathways and proteins in large stones (when comparing large stones vs sand), coagulation machinery and protease inhibitors are significantly increased. (B) There is also enrichment for coagulation-related pathways and hemoglobin binding and oxygen binding pathways.
Fig 5.
Pathway analysis of significantly changed proteins comparing large stones vs sand.
(A) Among downregulated pathways and proteins in large stones (when comparing large stones vs sand), RNA export, ribosomal binding and proteases are significantly decreased. (B) Polysome and ribosomal-related pathways are most significantly enriched, but there is also significant enrichment for chromosomal pathways. (C) Levels of many ribosomal proteins decrease drastically as ‘sand’ continues to grow into the large stones.
Fig 6.
Changes in diverse proteins detected displaying log-scale fold-changes.
The bar graphics all display a ratio (stone vs sand), or more specifically: log2-fold changes comparing either ‘small vs sand’, the comparison ‘medium vs sand’, and the comparison ‘large vs sand’ for all shown proteins. (A) Levels of prothrombin increased across all stone sizes (compared to sand), but most drastically when comparing large stones vs sand. (B) Plasminogen (PLG) shows the largest increase in small stones vs sand, and continues to show upregulation in medium/sand and large/sand. (C-D) Fibrinogen gamma-chain (FGG) shows large increases across all stone sizes when compared to sand, and beta chain (FGB) displays the largest increase in small stones vs sand, and continues to show upregulation in medium/sand and large/sand. (E-F) Lower levels of proteases Calpain 4 and Cathepsin D are found in large stones than in sand. (G) Protease inhibitor AHSG was increased in large stones vs sand. (H) SOD1 is most drastically changed in the large stones, compared to sand. (* = p<0.05; *** = p<0.0005).