Defining Cellular Injury in Diabetic Nephropathy by Single Cell RNA Sequencing
Despite almost universal implementation within the last 20 years of treatments that were presumed to be reno-protective, diabetes continues to rank as the #1 cause of ESRD. Diabetic nephropathy (DN) is characterized by glomerulopathy, albuminuria and progressive tubulointerstitial fibrosis. Understanding the precise transcriptional changes that occur in single podocytes, tubular epithelium and interstitial cells during diabetic nephropathy may allow us to infer novel cell states and heterogeneity among these cells that will inform our understanding of disease pathogenesis. Single cell RNA-sequencing (scRNA-seq) has a unique advantage in characterizing cell transcriptomes because it can detect them comprehensively on a genomic scale. We have successfully implemented scRNA-seq in human kidney tissue, but we are limited by two critical hurdles. First, our dissociation protocols selectively enrich for proximal tubule epithelia but lack podocyte, fibroblast and endothelial cells, precluding their study. Second, our microfluidic-based scRNA-seq protocol requires very fresh tissue and cannot be performed on archival material. In this application we will attempt to overcome these substantial barriers by performing single nucleus RNA-seq (sNuc-seq) on archival, fresh frozen human kidney tissue of known histology and diagnosis. We believe that the nuclear isolation protocol will free all nuclei within our frozen sample, not just proximal tubule nuclei as with our single cell dissociation experience to date. We have already identified two normal and two diabetic nephropathy kidney samples in collaboration with the Boston Nephrectomy Biobank, led by Sus Waikar MD, MPH. The ability to perfrom sNuc-seq on archival material would also represent an enormous advance and open many new opportunities to study human diabetic nephropathy. We predict that measuring the gene expression repertoire of single nuclei has tremendous power to reveal stochastic gene expression and unappreciated differences in cell states during diabetic nephropathy.