Snapshots of nascent RNA reveal cell- and stimulus-specific responses to acute kidney injury.

Tian Shen ,Edwin Mathieu,Sumit Mohan,Yuanji Li,Qingyin Zhang,Samuel K Sia,Fangming Lin,Chyuan-Sheng Lin,Jacob S Stevens,Run Banlengchit,Beatriz Desanti De Oliveira,Nicholas P Tatonetti,Andrew Beenken,Miriam Callahan,Jacob Stauber,Jonathan Barasch,Siddarth Arumugam,Katherine Xu,Alexandra Jacunski,Krzysztof Kiryluk,Neal Paragas,Nathaniel Askanase,Pamela I Good ,Tejashree S Gopal ,Maria V Grau ,Abraham Levitman ,Vivette D D’agati

doi:10.1172/jci.insight.146374

Tian Shen , Edwin Mathieu + Show 25 more

Open Access

https://doi.org/10.1172/jci.insight.146374

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Abstract

The current strategy to detect acute injury of kidney tubular cells relies on changes in serum levels of creatinine. Yet serum creatinine (sCr) is a marker of both functional and pathological processes and does not adequately assay tubular injury. In addition, sCr may require days to reach diagnostic thresholds, yet tubular cells respond with programs of damage and repair within minutes or hours. To detect acute responses to clinically relevant stimuli, we created mice expressing Rosa26-floxed-stop uracil phosphoribosyltransferase (Uprt) and inoculated 4-thiouracil (4-TU) to tag nascent RNA at selected time points. Cre-driven 4-TU–tagged RNA was isolated from intact kidneys and demonstrated that volume depletion and ischemia induced different genetic programs in collecting ducts and intercalated cells. Even lineage-related cell types expressed different genes in response to the 2 stressors. TU tagging also demonstrated the transient nature of the responses. Because we placed Uprt in the ubiquitously active Rosa26 locus, nascent RNAs from many cell types can be tagged in vivo and their roles interrogated under various conditions. In short, 4-TU labeling identifies stimulus-specific, cell-specific, and time-dependent acute responses that are otherwise difficult to detect with other technologies and are entirely obscured when sCr is the sole metric of kidney damage.

Highlights

Many different types of stimuli challenge the homeostatic functions of the kidney, and in some cases, damage its cells
We examined 2 human nephrectomies (~15 minutes warm ischemia) and 5 human biopsies with a range of pathological diagnoses, including rhabdomyolysis, tacrolimus toxicity, acute tubular injury (ATI) possibly secondary to urinary tract infection, and ischemic ATI in the setting of hepatorenal syndrome or in the setting of a nonsteroidal antiinflammatory agent (NSAIA)
Because we subcloned the uracil phosphoribosyltransferase (Uprt) enzyme into the Rosa26 locus, Uprt can be expressed in any cell of the mouse

Summary

Introduction

Many different types of stimuli challenge the homeostatic functions of the kidney, and in some cases, damage its cells. Serum creatinine (sCr) levels reflect the synthesis of creatinine by muscle cells and its clearance by filtration into the urine — a process described by the mass transfer equation, which yields the glomerular filtration rate. According to this equation, sCr levels reflect the excretory function of the kidney at steady state but not during periods when physiologic functions rapidly decline. It is not surprising that it is difficult to correlate sCr with histologic or molecular findings in humans and mice [10–14] These shortcomings affect real-time decision-making in pressured clinical settings, such as the emergency department [15]. A molecular approach to kidney injury might better align with etiologic information and with the patient’s clinical course, when molecular and physiologic markers are considered in combination [16–19]

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