Single cell RNA-seq on kidneys of mice with inactivated hepatocellular Mdm2.

In our study we aimed to characterise the effects of p53-induced liver senescence on murine kidneys. We induced liver senescence in 8-12 weeks old male mice by compromising hepatocellular Mdm2 functionality through systemic administration of the AAV8-TBG-Cre viral vector (ΔMdm2Hep mice). We observed transmision of the senescent phenotype from the senescent liver to the genetically unmanipulated kidney. To further elucidate the mechanisms that mediate this transmission phenotype and to characterise its effects on the kidney, we performed single-cell RNA sequencing (scRNA-seq) on the kidneys of 3 ΔMdm2Hep and 3 control mice. We observed the upregulation of several signalling pathways in specific renal cell compartments (e.g. TGFβ in the tubular compartment and JAK-STAT in the mesenchymal compartment). Our results show that liver senescence has a different effect on the renal transcriptome depending on the cellular compartment. This experiment contains 6 biological samples of kidney cells from mice with an AAV8-TBG-Cre that excises the floxed Mdm2 exons 5 and 6 in the liver (n=3 mice, Cre group) or AAV8-TBG-Null empty vector (n=3 mice) which does not affect the mentioned exons (control/Null group). 4 days post AAV injection the mice were culled and their kidneys were harvested. Cell viability and concentration were determined using the Trypan Blue assay. 20,000- 40,000 cells were loaded on a 10x Chromium chip (1 sample per lane). Cleanup, reverse transcription, cDNA amplification and library preparation were performed using the Chromium Single cell 3’ Reagent Kits (v3) as per the manufacturer’s instructions. The samples were sequenced in the Illumina NextSeq500 using the 2x150bp kit with the following read length parameters: 26bp Read1 - cell barcode and UMI, 8bp I7 index - sample index, 98bp Read2 - transcript read. Each biological sample has mutlple raw fastq files, corresponding to different reads or index (e.g. R1, R2, I1) and to different lanes used for sequencing (e.g. L001, L002, etc...).