Cell-specific translational profiling in acute kidney injury

Abstract

Acute kidney injury (AKI) promotes an abrupt loss of kidney function that results in substantial morbidity and mortality. Considerable effort has gone toward identification of diagnostic biomarkers and analysis of AKI-associated molecular events; however, most studies have adopted organ-wide approaches and have not elucidated the interplay among different cell types involved in AKI pathophysiology. To better characterize AKI-associated molecular and cellular events, we developed a mouse line that enables the identification of translational profiles in specific cell types. This strategy relies on CRE recombinase-dependent activation of an EGFP-tagged L10a ribosomal protein subunit, which allows translating ribosome affinity purification (TRAP) of mRNA populations in CRE-expressing cells. Combining this mouse line with cell type-specific CRE-driver lines, we identified distinct cellular responses in an ischemia reperfusion injury (IRI) model of AKI. Twenty-four hours following IRI, distinct translational signatures were identified in the nephron, kidney interstitial cell populations, vascular endothelium, and macrophages/monocytes. Furthermore, TRAP captured known IRI-associated markers, validating this approach. Biological function annotation, canonical pathway analysis, and in situ analysis of identified response genes provided insight into cell-specific injury signatures. Our study provides a deep, cell-based view of early injury-associated molecular events in AKI and documents a versatile, genetic tool to monitor cell-specific and temporal-specific biological processes in disease modeling.

Translational value of animal models of kidney failure

“…TRAP experiments studied mRNA expression during ischemia/reperfusion injury in 4 different lines of transgenic mice, each expressing the EGFP-L10a fusion protein in a different cellular compartment (kidney tubules, interstitial cells and glomerulus, endothelial cells, and immune cells). Each mouse line showed differential regulation during ischemia-reperfusion of a different set of genes, which was consistent with the function of each labeled cell (Liu et al, 2014). Necrosis, apoptosis, cell survival and cytoskeletal organization functions were increased in tubules.…”

Kidney Regeneration: Lessons from Development

“…We will discuss what is known and what is debated about the cellular mechanisms of repair after AKI. Then, taking advantage of recently published, cell-specific translational profiling studies performed in a mouse model of AKI 21 , we will provide a comprehensive analysis of the expression profiles of genes and pathways involved in embryonic kidney development that are activated after AKI, and discuss what published data is available to explain the functional roles of these genes in regenerative repair and tissue remodeling after AKI. We will also detail that while injury activates many of the same pathways involved in embryonic development, these are often activated in different cell types and lack the coordinated balance required for properly organized tissue regeneration and remodeling after injury.…”

Defining the Acute Kidney Injury and Repair Transcriptome

“…66 Here, TRAP relies on affinity purification of translating ribosomes through an eGFP-tagged, L10a ribosomal protein subunit (L10a::eGFP), and subsequent profiling of mRNAs stripped from the ribosome by microarray or RNA-seq (Figure 1). Cell type specificity is governed by the requirement for CRE-recombinase mediated removal of a transcription-blocking cassette upstream of an L10a::eGFP cDNA cassette: cell type specific CRE lines activate L10a::eGFP is distinct cell populations in the kidney (Figure 2).…”

“…In the recent study, distinct CRE lines enabled TRAP mRNA signatures to be generated for four critical cellular compartments in the kidney following IRI injury: the nephron, vascular, macrophage/monocyte and interstitial mesenchyme. 66 …”