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Single-cell RNA sequencing of murine kidneys for ischemia-reperfusion induced renal injury and fibrosis

ABSTRACT: Single-cell RNA sequencing of murine kidneys for ischemia-reperfusion induced renal injury and fibrosis

PROVIDER: PRJNA1148121 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR30238910_1.fastq.gz	Fastqsanger.gz
	SRR30238910_2.fastq.gz	Fastqsanger.gz
	SRR30238911_1.fastq.gz	Fastqsanger.gz
	SRR30238911_2.fastq.gz	Fastqsanger.gz
	SRR30238912_1.fastq.gz	Fastqsanger.gz

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Differential Ly6C Expression after Renal Ischemia-Reperfusion Identifies Unique Macrophage Populations

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Differential Ly6C Expression after Renal Ischemia-Reperfusion Identifies Unique Macrophage Populations

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Project description:Current methods to analyze gene expression measure steady-state levels of mRNA. In order to specifically analyze mRNA transcription, a technique has been developed that can be applied in-vivo in intact cells and animals. The technique is referred with the acronym NIAC-NTR (Non Invasive Application and Capture of Newly Transcribed RNA). This method makes use of the cellular pyrimidine salvage pathway and is based on affinity-chromatographic isolation of thiolated mRNA. When combined with data on mRNA steady-state levels, this method is able to assess the relative contributions of mRNA synthesis and degradation/stabilization. It overcomes limitations associated with currently available methods such as mechanistic intervention that disrupts cellular physiology, or the inability to apply the techniques in-vivo. The method was applied to study renal ischemia reperfusion injury, demonstrating its applicability for whole organs in-vivo. Affymetrix GeneChip microarrays were used to detail regulatory mechanisms of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level. Experiment Overall Design: The gene expression and regulation program at the RNA level of contralateral mouse kidneys of model IRI-mice was studied. Total RNA, amplified total RNA and specifically enriched newly transcribed RNA was isolated from normal untreated mouse kidneys and from contralateral mouse kidneys of IRI-mice. This experiment allowed detailed regulatory mechanisms in-vivo of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level of contralateral kidneys early (3h) after ischemia-reperfusion-injury..

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Expression data of kidneys from Wild-type (WT) or Mincle-deficient (KO) mouse subjected to renal ischemia-reperfusion injury.

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Project description:Chronic kidney disease is associated with progressive renal fibrosis, where perivascular cells give rise to the majority of Î±-SMA positive myofibroblasts. We sought to identify pericytic miRNAs that could serve as a target to decrease myofibroblast formation. We induced kidney fibrosis in FoxD1-GC;Z/Red-mice by unilateral ureteral obstruction (UUO) followed by FACS sorting of dsRed-positive FoxD1-derivative cells and miRNA profiling. MiR-132 selectively increased 21-fold during pericyte-to-myofibroblast formation whereas miR-132 was only 2.5-fold up in total kidney lysates (both in UUO and ischemia-reperfusion injury). MiR-132 silencing in UUO decreased collagen deposition (35%) and tubular apoptosis. Immunohistochemistry, western blot and qRT-PCR confirmed a similar decrease in interstitial Î±-SMA+ cells. Pathway analysis identified a rate-limiting role for miR-132 in myofibroblast proliferation that was confirmed in vitro. Indeed, antagomir-132 treated mice displayed a reduction in the number of proliferating, ki67+ interstitial myofibroblasts. Interestingly, this was selective for the interstitial compartment and did not impair the reparative proliferation of tubular epithelial cells, as evidenced by an increase in ki67+ epithelial cells, as well as increased (p-)RB1, Cyclin-A and decreased RASA1, p21 levels in kidney lysates. Taken together, silencing miR-132 counteracts the progression of renal fibrosis by selectively decreasing myofibroblast proliferation and could potentially serve as a novel antifibrotic therapy. Total RNA obtained from FACS sorted mouse FoxD1-derivative interstitial cells from healthy or fibrotic kidneys

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