Integration of spatial protein imaging and transcriptomics in the human kidney tracks the regenerative potential of proximal tubules

The organizational principles of nephronal segments are based on anatomical and physiological attributes that are linked to the homeostatic functions of the kidney. Recent molecular approaches have uncovered layers of deeper signatures and states in tubular cells that arise at various timepoints on the disease trajectory. Here, we introduce an analytical pipeline of multiplexed spatial protein imaging integrated with RNA expression to characterize proximal tubular subpopulations and neighborhoods in human kidney tissue. We demonstrate that, in reference tissue, a large proportion of S1 proximal tubular epithelial cells express THY1, a mesenchymal stromal and stem cell marker that regulates differentiation. Kidney disease is associated with loss of THY1 and transition towards expression of PROM1, another stem cell marker recently linked to failed repair. Our data support a model in which the interplay between THY1 and PROM1 expression in proximal tubules associates with their regenerative potential and marks the timeline of disease progression. The preparation and imaging of tissues were done using Visium Spatial Gene Expression protocol for polyA capture in fresh frozen tissue (10x Genomics, CG000240 protocol, Visium Tissue Preparation Guide). Using a Keyence BZ-X810 microscope equipped with a Nikon 10x CFI Plan Fluor objective, hematoxylin and eosin (H&E) stained brightfield mosaics were collected and stitched prior to mRNA capture. mRNA was extracted from tissue after 12 minutes of tissue permeabilization. Isolated mRNA was captured by oligonucleotides in the fiducial capture areas and reverse transcribed. Libraries were prepared based on the Visium 1.0 protocol (10X genomics CG000239 protocol) which included, second-strand synthesis, denaturation, cDNA amplification, and cleanup using SPRIselect (Beckman Coulter). Multiplexed sequencing was performed on a NovaSeq6000 (Illiumina). Sequencing data was demultiplexed and mapped to the reference genome GRCh38 3.0.0, and counted in Space Ranger (10X Genomics, v1.0.0). Data processing was performed in Seurat (v.4.4). The sample in this series is integrated with the samples GSM6047778 and GSM6047779 from GSE183456 (For GSM6047778 and GSM6047779, there has been no separate reanalysis or additional processing performed). *************************************************************** The table below lists GEO accessions reused for this study. ***************************************************************