The renal medulla is a specialized region of the kidney with important homeostatic functions. It has also been implicated in genetic and developmental disorders and ischemic and drug-induced injuries. Despite its role in kidney function and disease, the medulla’s baseline gene expression and epigenomic signatures have not been well described in the adult human kidney. Here we generate and analyze gene expression (RNA-seq), chromatin accessibility (ATAC-seq), chromatin conformation (Hi-C) and digital spatial profiling data from adult human kidney cortex and medulla. Using data from our carefully annotated specimens, we assign samples in the larger public GTEx database to cortex and medulla, thereby identifying several misassignments and extracting meaningful medullary gene expression signatures. Using integrated analysis of gene expression, chromatin accessibility and conformation profiles, we reveal insights into medulla development and function. Our datasets will also provide a valuable resource for researchers in the GWAS community for functional annotation of genetic variants.
Overall design
Human nephrectomy tissues were fixed in formalin and paraffin embedded (FFPE). Single tissue sections of kidney cortex and medulla from 3 different donors (RAM012, 013, 015) were applied to 3 SuperFrost charged slides. In preparation for digital spatial profiling, the slides were baked and then subjected to heat-induced antigen retrieval and proteinase K digestion (1µg/ml). Human whole transcriptome atlas probes were applied to each slide and incubated at 37°C overnight. The following day, after stringent washes, slides were blocked and then counterstained with SYTO13 (labels DNA) and the following 2 antibodies: Alexafluor-532 conjugated anti-pan-cytokeratin (labels some epithelial cells in kidney) and Alexafluor-647 conjugated anti-CD10 (labels glomerular podocytes and proximal tubule cells in kidney). The slides were then loaded into the Nanostring GeoMx DSP instrument and scanned at 20x magnification (exposure times: SYTO13, 100ms; pan-CK, 400ms; CD10, 400ms). Regions of interest (ROI) were selected and bound probes were captured by the instrument. The captured probes were converted into an Illumina NGS compatible library using the SeqCode A barcode kit from Nanostring. The library was QC'ed using an Agilent TapeStation and quantified using a Qubit fluorometer. The library was seqeunced using a NextSeq 2000 SP flowcell.
Less...
More...