Extracellular RNA moves from the glomerulus to the renal tubule

There is a wealth of indirect evidence that extracellular RNA (exRNA) signalling can regulate renal tubular epithelial cell function. However, the physiological importance of this signalling is uncertain. We sought to determine the extent of extracellular RNA transfer between cells in a healthy kidney. We tested the hypothesis that RNA travels from glomerular podocytes to renal tubular epithelial cells. We developed a method to track exRNA in the kidney using SLAMseq (SH-linked alkylation for the metabolic sequencing of RNA in tissue). We crossed podocin-Cre mice with floxed-stop-UPRT mice to express recombinant uracil phosphoribosyl transferase (UPRT) in podocytes. Mice were injected with the modified nucleobase 4-thiouracil, which is incorporated into nascent RNA with high efficiency only in UPRT-expressing cells. We harvested glomeruli or tubular cells, extracted RNA and prepared libraries for SLAMseq, in which sites of mRNA labelling with 4-thiouracil are detected as T>C conversions in 3UTRs. In glomeruli, we detected labelling of known podocyte genes but not of genes known to be restricted to endothelial, renal tubular or white blood cells. Setting a false-discovery rate of 1%, the proportion of genes deemed to be labelled with high confidence was 7.1% (95% confidence interval 6.8 - 7.4%) in 4TU-treated podocyte-UPRT mice, 2.5% (2.3 - 2.7%) in Cre-negative controls and 1.0% (0.9 - 1.1%) in 4TU-naive controls. In tubular cells, we detected a small but statistically significant increase in RNA labelling in podocyte-UPRT mice compared to Cre-negative controls (p = 7.4 x 10-16 in a zero-inflated Poisson regression model). We conclude that RNA is transferred from podocytes to renal tubular epithelial cells in vivo under physiological conditions. Our model provides the opportunity to explore the consequences of this novel signalling pathway in health and kidney disease.