Highly multiplexed immunofluorescence of the human kidney using co-detection by indexing

Abstract

The human kidney is composed of many cell types that vary in their abundance and distribution from normal to diseased organ. As these cell types perform unique and essential functions, it is important to confidently label each within a single tissue to accurately assess tissue architecture and microenvironments. Towards this goal, we demonstrate the use of co-detection by indexing (CODEX) multiplexed immunofluorescence for visualizing 23 antigens within the human kidney. Using CODEX, many of the major cell types and substructures, such as collecting ducts, glomeruli, and thick ascending limb, were visualized within a single tissue section. Of these antibodies, 19 were conjugated in-house, demonstrating the flexibility and utility of this approach for studying the human kidney using custom and commercially available antibodies. We performed a pilot study that compared both fresh frozen and formalin-fixed paraffin-embedded healthy non-neoplastic and diabetic nephropathy kidney tissues. The largest cellular differences between the two groups was observed in cells labeled with aquaporin 1, cytokeratin 7, and α-smooth muscle actin. Thus, our data show the power of CODEX multiplexed immunofluorescence for surveying the cellular diversity of the human kidney and the potential for applications within pathology, histology, and building anatomical atlases.

Keywords: CODEX; cell neighborhoods; cell type; immunofluorescence; microscopy; multiplexed imaging.

Figure 1:

A) CODEX multiplexed IF staining of a human kidney from a 66-year-old male. B) Enlargement of a region of the cortex with several glomeruli present as well as proximal tubules. C) Medullary region of the kidney with many tubules present. D&E) Enlargement of glomeruli surrounded by different tubular segments with high expression of α-smooth muscle actin and aquaporin 1, respectively. F) Tubules within the cortex with high expression of cytokeratin 7 and tryptase. G) Representative view of medulla. H) Portion of cortex with increased numbers of mast cells. I) Enlargement of medullary rays. The color legend for all panels is as follows: cytokeratin 7 (pink), tryptase (orange), nestin (yellow), ß-catenin (green), aquaporin 1 (teal), vimentin (dark blue). Scale bars are 1 mm for panel A, 200 μm for panels B–C, and 50 μm for panels D–I.

Figure 2:

A) Individual images of assayed antigens showing the diversity of structures within the kidney. B) Pearson’s correlation coefficients from three healthy kidney samples for glomerular markers, indicating cell types that are highly correlated (red), noncorrelated (white), and anti-correlated (blue). C) Correlation plot derived from three diabetic nephropathy kidneys, showing cellular redistribution resulting from disease. D) Similar correlation plot for tubular markers from healthy and E) diabetic nephropathy patients. Correlations were established between 0 and 40 μm from the center of the cell for the entire tissue and consist of ~1 million cells.