Dataset Information

A mouse model of renal fibrosis to overcome the technical variability in ischaemia/reperfusion injury among operators.

ABSTRACT: The ischaemia-reperfusion (I/R) model is a widely used model of acute kidney injury (AKI) and renal fibrosis. However, the ischaemia duration that is long enough to cause broad fibrosis shows that a high mortality rate and a short ischaemia duration does not cause fibrosis, resulting in a large variation in fibrosis progression in this experimental model. Inter-operator variation occurs for I/R injury severity because the I/R procedure is complex, which results in poor reproducibility of subsequent fibrosis in the model. In the present study, we developed a renal fibrosis model in which the fibrosis progression for 8 weeks is predictable within 8 days. Three operators independently performed I/R followed by uninephrectomy at day 7 in mice. The aim was to create a model that would show a blood urea nitrogen (BUN) level >100?mg/dL at day 8 after I/R (day 1 after uninephrectomy). Although the ischaemia duration to satisfy this BUN criterion differed among operators, the mice developed anaemia, polyuria, and fibrosis in a similar manner under the same BUN criterion with a low mortality rate. Interstitial fibrosis had developed at week 8, which was strongly correlated with the BUN at day 8. This protocol allows operators to adjust the ischaemia duration based on the BUN criterion and to separate mice into the desired number of groups based on the BUN to study interventions against renal fibrosis.

SUBMITTER: Guan Y

PROVIDER: S-EPMC6639321 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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A mouse model of renal fibrosis to overcome the technical variability in ischaemia/reperfusion injury among operators.

Guan Yu Y Nakano Daisuke D Zhang Yifan Y Li Lei L Tian Ye Y Nishiyama Akira A

Scientific reports 20190718 1

The ischaemia-reperfusion (I/R) model is a widely used model of acute kidney injury (AKI) and renal fibrosis. However, the ischaemia duration that is long enough to cause broad fibrosis shows that a high mortality rate and a short ischaemia duration does not cause fibrosis, resulting in a large variation in fibrosis progression in this experimental model. Inter-operator variation occurs for I/R injury severity because the I/R procedure is complex, which results in poor reproducibility of subsequ ...[more]

PMID: 31320707

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Project description:Acute kidney injury (AKI) is associated with an abrupt loss of kidney function that results in significant morbidity and mortality. Considerable effort has focused around the identification of diagnostic biomarkers and the analysis of molecular events. Most studies have adopted organ-wide approaches that do not fully capture the interplay among different cell types in the pathophysiology of AKI. To extend our understanding of molecular and cellular events in AKI, we developed a mouse line that enables the identification of translational profiles in specific cell types by CRE recombinase-dependent activation of an eGFP-tagged L10a ribosomal protein subunit, and consequently, translating ribosome affinity purification (TRAP) of mRNA populations. By utilizing cell-type specific CRE-driver lines, in this study we identify distinct cellular responses in an ischemia reperfusion injury (IRI) model of AKI. Cell-specific translational expression profiles were uncovered 24 hours after IRI from four populations enriched for distinct anatomical and cellular subgroups: nephron, interstitial cell populations, vascular endothelium, and macrophages/monocytes by Affymetrix microarray. A construct containing the CAGGS promoter driving eGFP-L10a, with a loxP-site flanked triple SV40 polyA cassette between promoter and eGFP-L10a cassette was targeted into the ubiquitously active Rosa26 locus. The upstream polyA cassette is designed to block activity of the downstream eGFP-L10a cassette. CRE-dependent removal of this transcriptional block activates eGFP::L10a production within the CRE-producing cell, and all of its descendants. Mice carrying the conditional eGFP-L10a allele, referred to as L10a, were maintained in a homozygous state. L10a mice were crossed to four CRE strains to activate eGFP::L10a expression in four predominantly non-overlapping cellular compartments in the kidney. A Six2-Tet-GFP::CRE allele is active exclusively within nephron progenitors; consequently, historical labeling results in eGFP::L10a expression throughout the main body of the nephron. A Foxd1-GFP::CRE allele is active in the progenitors of many of the interstitial cell lineages including those generating mesangial and non-glomerular pericytes. In addition, Foxd1 is normally expressed in podocytes. Cdh5-CRE is reported to be active throughout the vascular endothelium, and finally, Lyz2-CRE specifically labels cells of the myeloid lineage, notably macrophages, monocytes and dendritic cells. Mice carrying any CRE allele and the L10a allele are designated generically CRE-L10a. six2-L10a, foxd1-L10a, cdh5-L10a and lyz2-L10a denote specifically mice that are compound heterozygotes for the indicated CRE driver and L10a. CRE-L10a, L10a heterozygous littermates without CRE allele, C57BL/6 wild type mice were subjected to renal bilateral warm ischemia 28 minutes followed by 24-hour reperfusion when the kidney TRAP RNA and total RNA were isolated and subjected to Affymetrix microarray. Biological triplicates for each CRE-L10a line underwent no Surgery; sham Surgery and IRI treatment.

Dataset Information

A mouse model of renal fibrosis to overcome the technical variability in ischaemia/reperfusion injury among operators.

Publications

A mouse model of renal fibrosis to overcome the technical variability in ischaemia/reperfusion injury among operators.

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