Project description:INS1-EGFP-L10a stable cell lines were induced with Doxycycline (Dox) for 24 hours, then either untreated or treated for 30 minutes with 1uM Thapsigargin to induce ER stress. Total RNA was purified with Trizol, and RIP RNA samples were extracted by immunoaffinity purification using an EGFP antibody We aimed to determine which genes are enriched under ER stress at the polyribosomal level. To do this we compared expression profiles of Total RNA with and without ER stress, and Immunoaffinity purified RNA (RIP) with and without ER stress. Microarray results of INS-1 EGFP-L10a cells either untreated or treated with 1uM Thapsigargin (Tg) in triplicate. Both Total RNA and RNA isolated from ribosomal Immunoprecipitation (RIP) When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to cause programmed cell death. We discovered that thioredoxin-interacting protein (TXNIP) is a critical node in this M-bM-^@M-^\Terminal UPRM-bM-^@M-^]. TXNIP becomes rapidly induced by hyperactivated IRE1a, an ER bifunctional kinase/endoribonuclease (RNase). IRE1a controls TXNIP mRNA stability by reducing levels of a TXNIP destabilizing micro-RNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing Caspase-1 cleavage and interleukin 1b (IL-1b) secretion. Txnip gene deletion reduces pancreatic b-cell death during ER stress, and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule IRE1a RNase inhibitors suppress TXNIP production to block IL-1b secretion. In summary, the IRE1a-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death, and may be targeted to develop new treatments for degenerative diseases driven by ER stress. There are 12 samples total all in INS-1 EGFP L10a cells- 3 untreated total RNA (reference sample), 3 treated with 1uM Tg 30 min total RNA, 3 untreated RIP RNA (reference sample), 3 treated with 1uM for 30 min RIP RNA

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.

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