Spatial heterogeneity in gene expression changes associated with diabetic kidney disease in human kidneys. (A) Left: Representative fluorescence images of normal and diabetic human kidneys. Tissues were stained with antibodies against PanCK, WT1, and PTPRC. Right: Example images from a normal kidney highlighting the AOI strategy. Glomeruli were profiled using polygon-shaped areas of illuminations (AOIs), and tubules were automatically segmented into proximal tubules (PanCK−) and distal tubules (PanCK+). (B) Individual glomeruli in each kidney sample were annotated by degree of pathology. A representative H&E image (left) and fluorescence image (right) from the same region of a diabetic kidney disease (DKD) specimen are shown. Glomeruli with a higher degree of abnormality are circled in gray and labeled “A”, whereas more normal glomeruli are circled in white and labeled “N”. (C) Principal component analysis of variation between samples using genes detected above background in >1% of AOIs. PC1 versus PC2 is plotted, with substructure indicated by color and disease status indicated by shape. (D) Boxplots of counts in all AOIs of three example genes differentially expressed between kidney substructures with the corresponding antibody-stained images from the Human Protein Atlas (https://www.proteinatlas.org/) (Uhlén et al. 2015). (E) Left: Heatmap of differentially expressed genes between normal and DKD in glomeruli, distal tubules, and proximal tubules. All genes are significant at FDR < 0.05 and a fold change of >1.5. Genes are annotated by the structure in which they were significantly differentially expressed, or “multiple” for the genes significant in more than one structure. Columns and rows are clustered by hierarchical clustering and the data are scaled by row. Right: Boxplot of normalized counts for two example differentially expressed genes in normal and DKD kidney structures. (F) Left: Results of cell type deconvolution of glomeruli using single-cell expression data from Young et al. (2018). Data are displayed as stacked barplots with each bar as a single AOI and the estimated proportion of each cell type colored, faceted by disease status. Right: Boxplots of proportions of two example differentially abundant cell types in normal and DKD glomeruli (t-test Bonferroni-corrected P-value < 0.05). MNP, mononuclear phagocytes; DC, dendritic cells. (G) Pie charts overlaid on the fluorescence image of a single kidney, showing the proportion of different glomerulus and immune cell types for each glomerulus profiled in a representative disease sample. Each plot is outlined based on pathological annotation: abnormal glomeruli (blue), healthy glomeruli (red).
