PROJECT SUMMARY – Kidney OSP. The overall structure and anatomical relationships among different cell types within the human kidney are well understood, and experimental studies have defined the molecular and functional characteristics of individual kidney cell types. However, remarkably little is known about the integration, interactions, and molecular cross- talk between the different cellular compartments in normal kidneys. The goal of the Kidney OSP is to address this limitation in current knowledge by developing a fully integrated, multimodal molecular imaging pipeline to characterize the cellular and molecular organization of the human kidney. Building on our already established HuBMAP characterization pipeline, we will bring together imaging mass spectrometry, highly multiplexed immunofluorescence microscopy, autofluorescence microscopy, stained microscopy, spatial transcriptomics, spatial proteomics, and single-cell RNA-seq. Each modality was specifically chosen to enable the construction of a kidney molecular atlas that spans a wide range of spatial scales (e.g., single cells, functional tissue units, neighborhoods, and whole organs) and molecular classes (e.g., metabolites, lipids, proteins, and RNAs). The result will be a comprehensive molecular atlas of the human kidney that will be fully compatible with other molecular interrogation pipelines and data generated by HuBMAP researchers. In Aim 1 we will continue our kidney tissue collection and management protocols that build on our established kidney biobanks and tissue acquisition programs to create a representative molecular atlas of the human kidney. Aim 2 establishes clinical and histological standards and metrics that will be used to determine the suitability of samples that can be entered into the analytical pipeline. The focus of Aim 3 puts forth a strategy for 2-D and 3-D multimodal imaging and deep multi-omic analysis of the human kidney. Assay development on surgical resection specimens will be applied to whole organs using non-transplantable donor kidneys to construct 3-D molecular atlases. Finally, Aim 4 will scale up the kidney-specific characterization pipeline to generate representative 3-D molecular atlases from multiple, intact human kidneys.