Spatiotemporal transcriptomic analysis during cold ischemic injury to the murine kidney reveals compartment-specific changes

Kidney transplantation is the preferred treatment strategy for end-stage kidney disease. Deceased donor kidneys usually undergo cold storage until kidney transplantation, leading to cold ischemia injury that may contribute to poor graft outcomes. However, the molecular characterization of potential mechanisms of cold ischemia injury remains incomplete. To bridge this knowledge gap, we leveraged the 10x Visium spatial transcriptomic technology to perform full transcriptome profiling of murine kidneys subject to varying durations of cold ischemia typical in a deceased donor kidney transplant setting. We developed a computational workflow to identify and compare spatiotemporal transcriptomic changes that accompany the injury pathophysiology in a tissue compartment-specific manner. We identified proportional enrichment of oxidative phosphorylation (OXPHOS) genes with increasing duration of cold ischemia injury within the oxygen-lean inner medulla region, suggestive of atypical metabolic presentation. This was distinct in cold ischemia injury tissue compared to warm ischemia-reperfusion kidney injury tissue. Spatiotemporal trends were validated by qPCR and immunofluorescence in a larger cohort of mice. We provide an interactive online browser at https://jef.works/CellCarto-ColdIschemia/ to facilitate exploration of our results by the broader scientific and clinical community. Altogether, our spatiotemporal transcriptomic analysis identified coordinated molecular changes within metabolic pathways such as OXPHOS deep within the cold ischemic kidney, highlighting the need for increased attention to the inner medulla and potential opportunities for new insights beyond those available from superficial biopsy-focused tissue examinations. Here, we provide processed spatial transcriptomics data, metadata, and relevant annotations derived through our computational workflow as described in the main article. There are four compressed folders corresponding to different durations of cold ischemia injury (CIS) i.e., 0, 12, 24 and 48 hours called: 1_0hr.tar.gz 2_12hr.tar.gz 3_24hr.tar.gz 4_48hr.tar.gz Each of these compressed folders contain folders called: spatial filtered_feature_bc_matrix These folders are standard outputs from the 10x Genomics SpaceRanger (v2.0.1) pipeline. 1. The “spatial” subfolder folder contains the following files (descriptions from 10XGenomics): Filename Description aligned_fiducials.jpg Aligned fiducials QC image aligned_tissue_image.jpg Aligned CytAssist and Microscope QC image. Present only for CytAssist workflow cytassist_image.tiff Input CytAssist image in original resolution that can be used to rerun the pipeline. Present only for CytAssist workflow detected_tissue_image.jpg Detected tissue QC image scalefactors_json.json Scale conversion factors for spot diameter and coordinates at various image resolutions spatial_enrichment.csv Downsampled full resolution image. The image dimensions depend on the input image and slide version. tissue_hires_image.png Downsampled full resolution image. The image dimensions depend on the input image and slide version. tissue_lowres_image.png Full resolution image downsampled to 600 pixels on the longest dimension. tissue_positions.csv csv file containing spot barcode, if the spot was called under (1) or out (0) of tissue; the array position, image pixel position x, and image pixel position y for the full resolution image. 2. The “filtered_feature_bc_matrix” folder contains the following files: Filename Description barcodes.tsv.gz compressed file containing the barcode identity (16 nucleotide oligomer sequence) for each spot. features.tsv.gz compressed file containing gene names (features) along with their respective ensemble IDs. matrix.mtx.gz compressed sparse matrix file containing the raw gene counts information for each spot. Note: Count matrix (i.e., feature-barcode matrix) and position matrix can be constructed from these files using the code provided in the folder named 0.Count_&_Position_matrix_construction located within the following GitHub repository: https://github.com/ssingh95jhu/Cold_Ischemia_Injury_Molecular_Characterization_v2 3. The raw sequence files for the spatial transcriptomics data corresponding to the 0, 12, 24 and 48 hours of cold ischemia injury are provided in the BAM format and are called: 5_0hr_bam.bam 6_12hr_bam.bam 7_24hr_bam.bam 8_48hr_bam.bam 4. The "structure_annotation.csv.gz" file contains spot barcode identity and their corresponding annotation belonging to one of the kidney compartments namely inner medulla, outer medulla, cortex and other. These files were used for a web application to interactively explore this data. Spot barcode identities have been modified to substitute ‘-’ with ‘.’ but are otherwise the same.