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Inflammation primes the murine kidney for recovery by activating AZIN1 adenosine-to-inosine editing
Segewkal Hawaze Heruye, … , Pierre C. Dagher, Takashi Hato
Segewkal Hawaze Heruye, … , Pierre C. Dagher, Takashi Hato
Published July 2, 2024
Citation Information: J Clin Invest. 2024;134(17):e180117. https://doi.org/10.1172/JCI180117.
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Research Article Nephrology

Inflammation primes the murine kidney for recovery by activating AZIN1 adenosine-to-inosine editing

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Abstract

The progression of kidney disease varies among individuals, but a general methodology to quantify disease timelines is lacking. Particularly challenging is the task of determining the potential for recovery from acute kidney injury following various insults. Here, we report that quantitation of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing offers a distinct genome-wide signature, enabling the delineation of disease trajectories in the kidney. A well-defined murine model of endotoxemia permitted the identification of the origin and extent of A-to-I editing, along with temporally discrete signatures of double-stranded RNA stress and adenosine deaminase isoform switching. We found that A-to-I editing of antizyme inhibitor 1 (AZIN1), a positive regulator of polyamine biosynthesis, serves as a particularly useful temporal landmark during endotoxemia. Our data indicate that AZIN1 A-to-I editing, triggered by preceding inflammation, primes the kidney and activates endogenous recovery mechanisms. By comparing genetically modified human cell lines and mice locked in either A-to-I–edited or uneditable states, we uncovered that AZIN1 A-to-I editing not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis to drive the recovery phenotype. Our findings implicate that quantifying AZIN1 A-to-I editing could potentially identify individuals who have transitioned to an endogenous recovery phase. This phase would reflect their past inflammation and indicate their potential for future recovery.

Authors

Segewkal Hawaze Heruye, Jered Myslinski, Chao Zeng, Amy Zollman, Shinichi Makino, Azuma Nanamatsu, Quoseena Mir, Sarath Chandra Janga, Emma H. Doud, Michael T. Eadon, Bernhard Maier, Michiaki Hamada, Tuan M. Tran, Pierre C. Dagher, Takashi Hato

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Figure 3

Azin1 A-to-I–uneditable state hinders cell growth and limits glycolytic capacity.

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Azin1 A-to-I–uneditable state hinders cell growth and limits glycolytic ...
(A) Sanger sequencing chromatograms for wild-type (HEK293T; top), AZIN1 A-to-I–locked (middle), and AZIN1 A-to-I–uneditable homozygous cell lines (bottom). Homology-directed repair donor oligonucleotides used for CRISPR knockin are shown in Supplemental Figure 5A. (B) Western blotting for AZIN1 under indicated conditions (~70% confluence). (C) Determination of AZIN1 protein turnover under indicated conditions. Nascent protein synthesis was inhibited with 250 μg/mL cycloheximide. Arrow points to AZIN1. Bands below AZIN1 result from inhibition of proteasomal degradation with MG132. n = 2 biological replicates. (D) Real-time monitoring of cell growth for AZIN1 A-to-I–locked, uneditable, and wild-type cells. n = 3 independent experiments with n = 6 technical replicates for each experiment. *P < 0.05 at all time points for indicated conditions, except the stationary phase between AZIN1 A-to-I–locked and wild-type cells. Representative images are shown in Supplemental Figure 5C. (E) Polyribosome profiling of AZIN1 A-to-I–locked and uneditable cell lines. n = 3 independent experiments. Mean polysome/monosome ratios for A-to-I–locked and uneditable genotypes are 4.1 and 3.6, respectively. (F) Heatmap of the top 20 differentially expressed genes between AZIN1 A-to-I–locked and uneditable cell lines as determined by RNA-Seq (https://connect.posit.iu.edu/azin1/). (G) Cell growth under indicated conditions. Representative images are shown in Supplemental Figure 5D. *P < 0.05, **P < 0.05 after day 1 and day 2.5 for indicated conditions, respectively. (H) Extracellular acidification rates under indicated conditions (Seahorse glycolysis stress test). n = 3 independent experiments with n = 3 technical replicates for each experiment. *P < 0.05 vs. AZIN1-uneditable cells at indicated time points. (I) Identification of AZIN1-interacting molecules by mass spectrometry. Top: Coomassie staining for input, flow-through, and immunoprecipitated unfractionated lysates from IgG control and transfection of FLAG-tagged AZIN1 or AZIN1 without FLAG plasmids. Middle: Western blotting for AZIN1. Cells overexpressing FLAG-tagged A-to-I–locked AZIN1 or uneditable plasmids were fractionated into cytoplasmic and nuclear compartments and immunoprecipitated using anti-FLAG antibody (cytoplasmic fraction is shown). See also Supplemental Figure 5H. Summary of coprecipitated proteins with AZIN1 is presented in the bottom table. n = 3 independent experiments. *Plasmid construct not used in this article.

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