Hunter, R. W. | Search bioRxiv

bioRxiv2024

SLAMseq reveals transfer of RNA from liver to kidney in the mouse

Extracellular RNA (exRNA) mediates intercellular communication in plants and lower animals; whether it serves a signalling function in mammals is controversial. Reductionist experiments, in which a single RNA is over-expressed or tagged, have shown RNA transfer between tissues but these may not be relevant to normal physiology. For example, the microRNA miR-122 is released from injured hepatocytes and is taken up by kidney cells. We sought to determine the scale of RNA transfer between liver and kidney through the metabolic labelling of RNA in mice. We used 4-thiouracil to specifically label RNA in hepatocytes then detected labelled (thiolated) RNA in the kidney using SLAMseq: SH-Linked Alkylation for the Metabolic sequencing of RNA. In the kidney, mRNA labelling was detected in 5% of all kidney transcripts under healthy conditions and was increased to 34% of kidney transcripts after acute hepatocellular injury. Labelling was evident in kidney transcripts mapping to known hepatocyte marker genes, to a greater extent than those mapping to markers of other cell types. Labelled small RNA was not detected in kidney tissue. Our results are consistent with the transfer of RNA from liver to kidney; this transfer is augmented in liver injury.

molecular biology↗

bioRxiv2021

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.

molecular biology↗