BASIC RESEARCH www.jasn.org The Integrated RNA Landscape of Renal Preconditioning against Ischemia-Reperfusion Injury Marc Johnsen,1 Torsten Kubacki,1 Assa Yeroslaviz ,2 Martin Richard Späth,1 Jannis Mörsdorf,1 Heike Göbel,3 Katrin Bohl,1,4 Michael Ignarski,1,4 Caroline Meharg,5 Bianca Habermann,6 Janine Altmüller,7 Andreas Beyer,3,8 Thomas Benzing,1,3,8 Bernhard Schermer,1,3,8 Volker Burst,1 and Roman-Ulrich Müller 1,3,8 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Although AKI lacks effective therapeutic approaches, preventive strategies using precondi- tioning protocols, including caloric restriction and hypoxic preconditioning, have been shown to prevent injury in animal models. A better understanding of the molecular mechanisms that underlie the enhanced resistance to AKI conferred by such approaches is needed to facilitate clinical use. We hypothesized that these preconditioning strategies use similar pathways to augment cellular stress resistance. Methods To identify genes and pathways shared by caloric restriction and hypoxic preconditioning, we used RNA-sequencing transcriptome profiling to compare the transcriptional response with both modes of preconditioning in mice before and after renal ischemia-reperfusion injury. Results The gene expression signatures induced by both preconditioning strategies involve distinct com- mon genes and pathways that overlap significantly with the transcriptional changes observed after ischemia-reperfusion injury. These changes primarily affect oxidation-reduction processes and have a major effect on mitochondrial processes. We found that 16 of the genes differentially regulated by both modes of preconditioning were strongly correlated with clinical outcome; most of these genes had not previously been directly linked to AKI. Conclusions This comparative analysis of the gene expression signatures in preconditioning strategies shows overlapping patterns in caloric restriction and hypoxic preconditioning, pointing toward common molecular mechanisms. Our analysis identified a limited set of target genes not previously known to be associated with AKI; further study of their potential to provide the basis for novel preventive strategies is warranted. To allow for optimal interactive usability of the data by the kidney research community, we provide an online interface for user-defined interrogation of the gene expression datasets (http://shiny. cecad.uni-koeln.de:3838/IRaP/). JASN 31: 716–730, 2020. doi: https://doi.org/10.1681/ASN.2019050534 Received May 27, 2019. Accepted January 5, 2020. The incidence of AKI is steadily increasing, leading M.J., T.K., V.B., and R.-U.M. contributed equally to this work. to relevant morbidity and mortality and causing a growing economic burden to Western health care Published online ahead of print. Publication date available at systems.1 Despite extensive research, therapies for www.jasn.org. AKI are still lacking in clinical practice. In contrast, Correspondence: Dr. Roman-Ulrich Müller, Cologne Excellence preventive strategies using various so-called pre- Cluster on Cellular Stress Responses in Aging-Associated Dis- eases, Josef-Stelzmann-Street 26, 50931 Cologne, Germany, or conditioning protocols, including caloric restric- Dr. Volker Burst, Department II of Internal Medicine, University of tion (CR) and interventions activating hypoxia Cologne, Kerpener Street 62, 50937 Cologne, Germany. E-mail: signaling (ischemic preconditioning [IP] and hyp- [email protected] or [email protected] – oxic preconditioning [HP]),2 4 have been found to Copyright © 2020 by the American Society of Nephrology 716 ISSN : 1046-6673/3104-716 JASN 31: 716–730, 2020 www.jasn.org BASIC RESEARCH be extraordinarily effective in animal models. Although it has Significance Statement longbeenknownthatCR5 and activation of hypoxia signal- ing6,7 lead to an extension of healthy lifespan, more recent Preconditioning strategies, such as caloric restriction and hypoxic experiments demonstrated that a short-term application of preconditioning, show strongly protective effects in animal models CR or IP/HP induces robust protection of various organs, of AKI, and researchers hope exploration of these strategies might provide insights into translating these powerful interventions to the 8 9,10 9 including heart, kidney, brain, and liver. clinical setting. However, the molecular mechanisms underlying the So far, a comprehensive understanding of the molecular beneficial effects of short-term application of caloric restriction and mechanisms underlying the beneficial effect of short-term hypoxic preconditioning have remained elusive. The authors used CR and oxygen deficiency is still lacking. Strikingly, these RNA-sequencing transcriptome profiling to compare the tran- effects can be observed in a wide range of species from inver- scriptional response with both modes of preconditioning before and after renal ischemia-reperfusion injury, identifying genes and 10–13 tebrates to mammals, and the protective effect is not pathways commonly shared by the two strategies. A comparison of confined to distinct organs but rather affects whole organisms. these findings with genes dysregulated during AKI points to genes This strongly points to evolutionary conserved mechanisms involved in preconditioning-associated organ protection that can that are paramount for the cellular defense against various now be examined as potential therapeutic targets in AKI. injuries.5 The fact that CR and HP share their protective potential has day, respectively. No increase in mortality or morbidity was led us to hypothesize that they utilize common pathways. observed after HP. Here, we report our findings of a detailed comparative expres- sion analysis in kidneys of preconditioned mice. Renal Ischemia-Reperfusion Injury Model A warm renal ischemia-reperfusion injury (IRI) model was used as described elsewhere, with slight modifications.12 METHODS Briefly, after anesthesia with intraperitoneal application of ketamine/xylazine, the right kidney was removed and the Ethical Statement left renal pedicle was clamped for 40 minutes. Postsurgical All animal procedures were conducted in accordance with Eu- recovery was assessed on the basis of weight loss, activity (nest- ropean (European Union directive 86/609/EEC), national and ing, flight behavior, movement), and appearance (grooming, institutional guidelines and approved by local governmental tachypnoea, dehydration) on a daily basis. Mice were eutha- authorities (LANUV 84–02.04.2013.A158). nized at 4, 24, or 72 hours after ischemia. Animals Male C57BL6/J mice aged 8–12 weeks were housed under Analysis of Renal Function and Overall Health identical specific-pathogen-free conditions in group cages Performance (five animals per cage) at a relative humidity of 50%–60% Blood samples were collected via puncture of a buccal vein or fi and with a 12-hour light/dark rhythm. All mice received water by nal bleeding. Serum urea and creatinine levels were mea- ad libitum and all except the caloric restricted mice received sured on a Cobas C 702 and Creatinine Plus-Test version 2 food ad libitum.Foodwasobtainedfromssniff(Art. (both Roche Diagnostics, Mannheim, Germany). Mortality V1534–703; Soest, Germany). and a postoperative recovery score (see Supplemental Appendix 1) were assessed over a period of 72 hours after IRI. CR Average food intake was determined by daily weighing of re- Histopathology maining food pellets for a period of 2 weeks. For experiments, Acute tubular damage was evaluated in a blinded fashion by an CR was applied for 4 weeks and mice were fed 70% of observed experienced renal pathologist (H.G.) using sections stained average food consumption. Mice were weighed on a weekly with periodic acid–Schiff (five visual fields per section) and basis to monitor weight loss. Neither increased mortality nor categorized using the scoring system proposed by Tirapelli and morbidity were observed during CR. Goujon14,15 on the basis of the presence of vacuolization, ep- ithelial flattening, loss of brush border, loss of nuclei, and – HP necrosis. Results were graded 0 4 according to the affected For HP, mice were put in a sealed chamber with free access to area (1: 0%–25%, 2: 25%–50%, 3: 50%–75%, 4: 75%–100%). water and food on three consecutive days. To achieve a nor- mobaric, hypoxic environment, oxygen was gradually re- Terminal Deoxynucleotidyl Transferase–Mediated placed by nitrogen over a period of 20 minutes, yielding Digoxigenin-Deoxyuridine Nick-End Labeling Staining a final oxygen concentration of 8% (Oxygen sensor, Grei- DeadEnd Fluorometric TUNEL System (Promega) was used singer GMH 3690; GMH Meßtechnik GmbH, Remscheid, on formalin-fixed paraffinsections(2mm) according to the Germany). The animals were exposed to hypoxia for 2 hours manufacturer’s protocol. Pictures were taken with a Zeiss on the first day and 4 and 8 hours on the second and third Meta 710 confocal microscope for documentation. JASN 31: 716–730, 2020 Gene Expression Signature of Renal Organoprotection 717 BASIC RESEARCH www.jasn.org mRNA and MicroRNA Sequencing mitoXplorer was used to analyze and visualize mitochon- RNeasy mini Kit (Qiagen, Hilden, Germany) was used to iso- drial expression dynamics.24 For visualization of the data, late total RNA from snap-frozen kidneys. After removal of access http://mitoxplorer.ibdm.univ-mrs.fr/, then go to ribosomal RNA using biotinylated target-specific oligos com- “analysis,” choose “Mouse” as