Spatially-Preserved Protein and mRNA Expression-profiling of the human kidney
The human kidney is comprised of hundreds of thousands of nephrons, each of which functions semi-autonomously as its own organoid. Because the three dimensional architecture of each nephron is key to its function, any characterization of the cell types in the human kidney must occur in a spatially-preserved context. Imaging mass cytometry (IMC) is a modification of CyTOF that uses mass spec detection of heavy metal tagged antibodies to provide simultaneous localization and quantitation of up to 42 probes at 1µm resolution on the same tissue section. We have developed an IMC protocol for profiling protein expression and activation states in cells of normal human kidney sections. However, this approach remains limited by the small numbers of highly specific antibodies for human kidney cell-specific antigens and therefore cannot be rapidly expanded to investigate new targets. In the current proposal, we will adapt PLAYR (proximity ligation assay for RNA) for use in IMC to enable us to simultaneously quantify protein (using our pre-validated antibody mixes) and mRNA expression (using heavy metal conjugated cDNA mixes) within each cell of the normal and diabetic human kidney in the context of their spatial relationship to adjacent tubular, glomerular and interstitial cells.

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