Description
Single cell sequencing studies have characterized the transcriptomic signature of cell types within the kidney. However, the spatial distribution of acute kidney injury (AKI) is regional and affects cells heterogeneously. We first optimized coordination of spatial transcriptomics and single nuclear sequencing datasets, mapping 30 dominant cell types to a human nephrectomy. The predicted cell type spots corresponded with the underlying histopathology. To study the implications of AKI on transcript expression, we then characterized the spatial transcriptomic signature of two murine AKI models: ischemia reperfusion injury (IRI) and cecal ligation puncture (CLP). Localized regions of reduced overall expression were associated with injury pathways. Using single cell sequencing, we deconvoluted the signature of each spatial transcriptomic spot, identifying patterns of colocalization between immune and epithelial cells. Neutrophils infiltrated the renal medulla in the ischemia model. Atf3 was identified as a chemotactic factor in S3 proximal tubules. In the CLP model, infiltrating macrophages dominated the outer cortical signature and Mdk was identified as a corresponding chemotactic factor. The regional distribution of these immune cells was validated with multiplexed CO-Detection by inDEXing (CODEX) immunofluorescence. Spatial transcriptomic sequencing complements single cell sequencing by uncovering mechanisms driving immune cell infiltration and detection of relevant cell subpopulations.
Overall Design
A single human reference nephrectomy (female aged 59 without histologic evidence of kidney disease) was acquired from the Biopsy Biobank Cohort of Indiana (BBCI). From age-matched 8-10 week old 129/ SvEv mice (Taconic Biosciences, Albany, New York), tissues were acquired from a sham mouse in which the abdomen was opened and sutured back and mice which underwent ischemia-reperfusion injury (IRI) or cecal ligation and puncture (CLP). In the IRI model both renal pedicles were exposed and clamped for 22 minutes through a midline incision then released. In the CLP model, the cecum was ligated and punctured with a 25 gauge needle. Kidneys were excised upon sacrifice 6 hours after each procedure and frozen in Optimal Cutting Temperature (OCT) compound. Slide preparation (CG000240_Demonstrated_Protocol_VisiumSpatialProtocols_TissuePreparationGuide_Rev_A, 10X Genomics), imaging (CG000241_VisiumImagingGuidelinesTN_Rev_A, 10X Genomics), mRNA extraction, library preparation, and sequencing (CG000239_VisiumSpatialGeneExpression_UserGuide_RevD, 10X Genomics) were conducted according to Visium Spatial Gene Expression protocols . Frozen transverse 10 m sections from the human nephrectomy or each murine model were placed within the etched frames of the capture areas on the active surface of the Visium Spatial Slide. Tissue sections were fixed in methanol and stained with hematoxylin and eosin (H&E). Stained tissue sections were permeabilized for 12 minutes, and the cDNA libraries were prepared and sequenced on an Illumina NovaSeq 6000 with 28bp+120bp paired-end sequencing mode.
Curator
xm_li