Project title
利用細胞專一性轉譯特徵及全基因轉錄分析研究急性腎損傷中Cyr61的訊號傳導 (III)
Internal ID
MOST107-2314-B002-177-MY3
Principal Investigator
Start Date
August 1, 2020
End Date
July 31, 2021
Investigators
Organizations
Partner Organizations
Cell-Specific Translational Profiling and Genome-Wide Transcriptional Analysis of Cyr61 Signaling during Acute Kidney Injury (III) = 利用細胞專一性轉譯特徵及全基因轉錄分析研究急性腎損傷中Cyr61的訊號傳導 (III)
Keywords
acute kidney injury
binding sites
gene expression profiling
fibrosis
sequence analysis
Description
Acute kidney injury (AKI) is serious global healthy issue because of its increasing prevalence and associated high morbidity and mortality. Recent studies indicated that AKI also leads to the increased risk of chronic kidney disease and is associated with long-term adverse outcomes. Understanding the pathophysiology of AKI progression and identifying modifiable key factors are warranting. However, current studies addressing this issue are limited by the kidney’s complexity structures and cellular diversity. Cysteine-rich protein 61 (Cyr61) has been shown to play an important role in acute and chronic kidney disease. It links tubular epithelium injury with inflammation, maladaptive repair, capillary rarefaction, and progressive kidney fibrosis. Taking the advantages of gene sequencing technologies and genetic modifying methods, this project aims to use translating ribosome affinity purification (TRAP)-RNA sequencing to systemically identify important Cyr61-regulated genes expression and transcription factors in a cell-specific manner. Furthermore, we will use identified transcription factors for chromatin immunoprecipitation with high throughput DNA sequencing (ChIP-Seq) to genome-wide screening downstream targets. In the first year, well will generate Six2GCE/wt;Cyr61Flox/Flox;Rosa26EGFP-L10a/wt trigenic mice to conditionally knock out Cyr61 coupled and producing EGFP-tagged L10a ribosomal protein subunit only in renal tubular epithelial cells of mice kidneys. By using anti-GFP antibodies-coupled magnetic beads, we will perform TRAP-RNA sequencing to elucidate Cyr61 downstream signaling within injured renal tubular cells during AKI. In the second year, will generate Csf1rCreEsr1/wt;Rosa26EGFP-L10a/wt bigenic mice and target kidney macrophages after AKI with or without anti-Cyr61 antibody treatment. TRAP-RNA sequencing of the bound mRNA will allow us to investigate renal tubule-macrophage interaction during AKI. These gene expression profiles change will be comprehensively investigated by bioinformatics analysis and validated by in situ hybridization, immunohistochemistry, and flow cytometry. Based on these result, we plan to identify find important transcription factors that differentially expressed by the effect of Cyr61. In the third year, we will do chromatin immunoprecipitation by antibodies against selected transcription factors in cultured renal tubular epithelia cells and macrophages, then send samples for high-throughput DNA sequencing. The novelty of this 3-year project is our cell-specific, systemic and unbiased genetic approach to identify important Cyr61-meidated signal pathways during AKI. This project also introduces the new generation sequencing technologies in a cell-specific way to the AKI research filed and participating researchers. In the era of precision medicine, leveraging these novel technologies gives insights into the patient-centered therapy.