Exogenous Gene Transmission of Isocitrate Dehydrogenase 2 Mimics Ischemic Preconditioning Protection

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Alexander L. Kolb,1,2 Peter R. Corridon ,3 Shijun Zhang,1 Weimin Xu,3 Frank A. Witzmann,4 Jason A. Collett,4 George J. Rhodes,3 Seth Winfree,3,5 Devin Bready,1,3 Zechariah J. Pfeffenberger,3 Jeremy M. Pomerantz,3 Takashi Hato,3 Glenn T. Nagami,6,7 Bruce A. Molitoris ,3,5 David P. Basile,4 Simon J. Atkinson,1,3 and Robert L. Bacallao2,3

1Department of Biology, Indiana University–Purdue University, Indianapolis, Indianapolis, Indiana; 2Research Division, Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana; 3Division of Nephrology, 4Department of Cellular and Integrative Physiology, and 5Indiana Center for Biological Microscopy, Indiana University School of Medicine, Indianapolis, Indiana; 6Division of Nephrology, Department of Medicine, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California; and 7Department of Medicine, David Geffen School of Medicine at the University of California, Los Angeles Veterans Affairs Medical Center, Los Angeles, California

ABSTRACT Ischemic preconditioning confers organ-wide protection against subsequent ischemic stress. A substantial body of evidence underscores the importance of mitochondria adaptation as a critical component of cell protection from ischemia. To identify changes in mitochondria protein expression in response to ischemic preconditioning, we isolated mitochondria from ischemic preconditioned kidneys and sham-treated kidneys as a basis for comparison. The proteomic screen identiﬁed highly upregulated proteins, including NADP +-dependent isocitrate dehydrogenase 2 (IDH2), and we conﬁrmed the ability of this protein to confer cellular protection from injury in murine S3 proximal tubule cells subjected to hypoxia. To further evaluate the role of IDH2 in cell protection, we performed detailed analysis of the effects of Idh2 gene delivery on kidney suscep- tibility to ischemia-reperfusion injury. Gene delivery of IDH2 before injury attenuated the injury-induced rise in serum creatinine (P,0.05) observed in controls and increased the mitochondria membrane potential (P,0.05), maximal respiratory capacity (P,0.05), and intracellular ATP levels (P,0.05) above those in controls. This communication shows that gene delivery of Idh2 can confer organ-wide protection against subsequent ischemia- reperfusion injury and mimics ischemic preconditioning.

J Am Soc Nephrol 29: 1154–1164, 2018. doi: https://doi.org/10.1681/ASN.2017060675

Ischemic preconditioning (IPC) was ﬁrst observed observation that applying nonlethal doses of ische- by Murry et al.1 in 1986. Since the original mia to target organs can confer protection against subsequent injury, it has been thought that a full understanding of this phenomenon would offer an Received June 22, 2017. Accepted December 11, 2017. entree to therapy for ischemic injury. After the Published online ahead of print. Publication date available at demonstration by Murry et al.1 of this phenome- www.jasn.org. non, studies were conducted on different organs, Correspondence: Dr. Robert L. Bacallao, Research II, Suite E202, such as heart, brain, liver, and kidney to name a 950 West Walnut Street, Indianapolis, IN 46202-5188. Email: few.1–5 These studies conﬁrmed the work by Murry [email protected]

1154 ISSN : 1046-6673/2904-1154 JAmSocNephrol29: 1154–1164, 2018 www.jasn.org BASIC RESEARCH et al.1 and extended the observations to other organs. Significance Statement IPC in the kidney was first studied by Zager and Baltes.6 This work laid the foundation for studying IPC in the kidney. Using Ischemic preconditioning is a physiologic adaptation to a transient this foundation, Joo et al.7 showed that IPC consists of two ischemic event that confers protection against subsequent ischemic protective windows. These protective windows involve an injury. Depending on the organ under study and the method of ischemic preconditioning, the adaptive response to ischemia is acute window, which begins immediately after IPC and ex- variable. Although some of the signaling pathways responsible for tends for several hours, and a late window, which begins ischemic preconditioning have been identified, the mechanism of around 24 hours and lasts up to several weeks after recovery cellular adaptation remains incompletely understood. In this com- from injury. Acute IPC may be mediated by mechanisms re- munication, we show that delivering plasmids encoding isocitrate lated to ion channels and cell signaling, such as the reperfusion dehydrogenase 2 to kidneys confers resistance to subsequent ischemic injury and increases states 2 and 3 mitochondria respiration. injury salvage kinase and survivor activating factor enhance- These are similar changes to those observed in mitochondria iso- ment signaling pathways.8,9 The late phase involves transcrip- lated from ischemic preconditioned kidneys. This work shows that it tional activation and translation of cytoprotective proteins, is possible to use gene delivery to confer resistance to subsequent such as hypoxia-induced transcription factors or heat shock ischemic injury. proteins.10 Because mitochondria impairment represents an early step in the pathogenesis of renal injury, IPC may be serum creatinine back to levels in sham-operated controls. As mediated in part by preserving mitochondria function in re- expected, this maneuver significantly attenuated the elevation sponse to ischemia-reperfusion injury (IRI) by preserving cel- in serum creatinine in rats 24 hours after a second ischemic lular ATP levels, reducing reactive oxygen species (ROS) pro- insult (Figure 1A). At this time, kidney histology shows little 11 duction, and inhibiting cell death pathways. evidence of injury (Figure 1, B and C). Renal cortical mito- Therefore, we hypothesize that alterations in mitochondria chondria were isolated from IPC or sham IPC rats at 14 days protein content are a cellular adaptation that confers protec- and used for a proteomic screen. Table 1 shows the proteins tion observed in the IPC long-term window. To determine that are significantly altered in IPC rat kidney versus sham IPC alterations in mitochondria protein content during IPC, pro- rat kidney. Twelve proteins were significantly altered (six in- teomic analysis was used to identify potential candidates for creased and six decreased) by the 14-day IPC protocol (Table mediating long-term IPC effects. One candidate protein, iso- 1). Of these, IDH2 was chosen for further analysis because of citrate dehydrogenase 2 (IDH2), which is upregulated during its function in the TCA cycle and as a branch point enzyme for 12 IPC, is involved in the citric acid cycle and ROS reduction. lipid and protein syntheses. To confirm these findings, we re- Mutated forms of IDH2 and more frequently, IDH1 have been peated the IPC protocol and conducted immune blots for fi identi ed in approximately 80% of low-grade gliomas and IDH2 on mitochondria isolated from renal cortex, showing 13,14 secondary gliomas. IDH2 mutations have also been iden- an approximate tenfold increase in IDH2 compared with con- fi ti ed in acute myeloid leukemia, making up greater than one trol rats (Figure 1D). half of the IDH mutations described.15 Analysis of tumor se- quencing data in both glioma and acute myeloid leukemia revealed neomorphic mutations in IDH1 within the catalytic Idh2 Confers Protection against Hypoxia Injury site at residue R132 and IDH within the catalytic site at resi- Initial screening of Idh2 activity used immortalized murine dues R140 and R172.14,16 When these mutations are present, renal proximal tubule S3 cells (MPTCs), which were derived IDH synthesizes the oncometabolite (R)-2-Hydroxygluarate from a large T antigen mouse through direct microscopic dis- by reducing a-ketoglutarate from isocitrate.15 (R)-2-Hydrox- section and have been used in studies of acute ischemic injury ygluarate is associated with hypermethylation of CpG islands and nephrotoxic kidney injury.20,21 Toconfirm that exogenous throughout the genome17 and affects histone methylation.18 Idh2 targets mitochondria, in vitro transfection was performed Because these oncogenic mutations are dissimilar to the nor- using an N-terminal myc-tagged Idh2. Multiphoton imaging mal IDH2 cDNA and because our prior work in gene delivery revealed that myc-tagged Idh2 clearly localized in mitochon- showed gene expression lasting predominantly for 30 days, dria stained with Mitotracker red (Figure 1, E–I). The Man- this candidate protein was selected for further investigation ders coefficient for colocalization was 0.8, indicating a high using both in vitro and in vivo models of cellular injury to degree of mitochondria colocalization.22 S3 cells transfected evaluate whether this protein may mediate protection against with plasmids bearing Idh2 were resistant to necrosis after IRI.19 hypoxia compared with mock transfected cells (9%67.8% versus 42%627.6%; P,0.05) (Figure 1J). Cells transfected with Idh2 maintained higher intracellular ATP/AMP ratios RESULTS even in response to hypoxia (Figure 2A) and have elevated mitochondria membrane potential (Figure 2B). These in- Proteomic Analyses of IPC Mitochondria creases in membrane potential are also linked with improved IPC was induced by subjecting rats to bilateral renal IRI for 30 cell health and survival (P,0.05) (Figures 1H and 2B). minutes and recovery for 14 days, resulting in the resolution of

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Figure 1. IPC or Idh2 protects kidney from injury. (A) Rats were subjected to sham, sham IPC IRI, and IPC IRI surgeries and subsequent IRI evaluated by serum creatinine (SCr) after 24 hours (n=5 per group; P,0.05 sham IPC versus IPC). *P,0.05. Hematoxylin and eosin– stained sections of (B) renal cortex and (C) outer medulla in sham IPC animals. Scale bar, 20 mm. (D) Immune blot of IDH2 in animals injected with saline or Idh2 or after IPC (left panel). Right panel illustrates the ratio of IDH2 to Actin using FIJI (n=6 per group). *P,0.05, IDH2 versus vehicle; #P,0.05, IPC versus vehicle. IDH2 myc localization in (I) National Institutes of Health 3T3 cells transfected with N-terminal myc-tagged Idh2 or (E) cells that were not transfected. (F) Blue channel for nuclei stained with Hoescht 33342. (G) Green channel for anti-myc labeled with FITC. (H) Red channel showing Mitotracker staining. Scale bar, 20 mm. (J) Quantitative analysis of PI staining of S3 MPTCs. All bar graphs are expressed as mean6SD. *P,0.05, hypoxic Idh2 versus hypoxic control.

Figure 3A depicts the effects of Idh2 transfection on mito- Idh2 transfection (Figure 3C). Complex 2 OCR, evaluated chondria respiration in S3 cells. Baseline oxygen consumption after the addition of Rotenone and Antimycin A, was also rate (OCR) of MPTCs was 29.466.8 pmol/min per cell, significantly increased by either myc-Idh2 or Idh2 transfec- whereas transfection with Idh2 or myc-tagged Idh2 signifi- tion (Figure 3D). Finally, in vitro analysis was to test if Idh2 cantly increased the OCR to 4162 and 39.664.7 pmol/min over expression affected reserve or spare respiratory capacity per cell, respectively (Figure 3B). Transfection with (SRC). This is measured as the difference between maximal either Idh2 or myc-tagged Idh2 resulted in a statistically sig- respiration and baseline respiration. Control S3 cell SRC was nificant increase in OCR compared with control or mock 23.568.4 pmol/min per cell. In cells transfected with Idh2, transfected cells (Figure 3B). Maximal uncoupled respiratory SRC was 31.9611 pmol/min per cell, indicating that the ad- capacity after addition of carbonyl cyanide p-trifluorome- dition of Idh2 increased MPTC SRC. However, the SRC in thoxyphenylhydrazone was also enhanced by Idh2 or myc- myc-Idh2 transfected cells was 26.967.8 pmol/min per cell, a

Table 1. Proteomic analysis of mitochondria isolated from IPC or sham IPC kidney cortex Gene Symbol Gene Name Fold Difference SULT1C2 SULFOTRANSFERASE 1C2 LIKE 2.4 IDH2 ISOCITRATE DEHYDROGENASE (NADP+) MITOCHONDRIAL 2.2 HIST1H2BC HISTONE H2B 2.0 C1QBP COMPLEMENT COMPONENT1Q SUBCOMPONENT BINDING PROTEIN 1.9 ACSL1 LONG-CHAIN FATTY ACID COA LIGASE 1 1.7 ACOT2 ACYL-COENZYME ATHIOESTERASE 2, MITOCHONDRIAL 1.5 THUMPD2 THUMPD DOMAIN CONTAINING 2 ISOFORM 1 23.0 MTCH2 MITOCHONDRIAL CARRIER HOMOLOG 2 21.9 HSDL2 HYDROXYSTEROID DEHYDROGENASE-LIKE PROTEIN 2 21.6 SLC25A13 SOLUTE CARRIER FAMILY 25, MEMBER 13 21.6 NDUFA5 NADH DEHYDROGENASE (UBIQUINONE) 1a SUBCOMPLEX SUBUNIT 5 21.6 SUCLG1 SUCCINATE COA LIGASE, a-SUBUNIT 21.6

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Gene Delivered IDH2 Targets to Mitochondria Retrograde hydrodynamic injection of plasmid cDNA through the renal vein has been shown to be an effective means of facilitat- ing exogenous gene expression in the rat kidney.19 To evaluate the effects of Idh2 up- regulation in the kidney, N-terminal myc- tagged Idh2 or saline was delivered by hydrodynamic injection. After 7 days of recovery, isolated renal cortical mitochon- Figure 2. Idh2 transfection alters mitochondria activity and function. (A) ATP-to-AMP dria contained approximately threefold ratios in MPTCs with or without Idh2 and hypoxia. (B) JC-1 fluorescent intensity ratios higher levels of IDH2 versus animals in- in MPTCs with or without Idh2 after hypoxia. All bar graphs are expressed as mean6 jected with saline alone as indicated by im- SD. *P,0.05, hypoxic Idh2 versus hypoxic control; #P,0.05, IDH2 versus control. mune blots (Figure 4A). These data indicate that hydrodynamic delivery of Idh2 results in sustained expression of protein value that was not significantly different from mock or con- in proximal tubule mitochondria to levels comparable with trol samples. This likely reflects a partial inhibitory effect of those observed during resistance to IRI after IPC. the myc tag on IDH2 integration in the oxidative phosphorylation pathway. IDH2 Transfection In Vivo and IPC Lead to Changes in Mitochondria Polarization Because IDH2 was found to localize to mitochondria, we analyzed changes in mitochondria function in response to IRI.

Figure 3. IDH2 increases cellular respiration in MPTCs. (A) Respiration was analyzed using a Seahorse. Seven micromolar carbonyl cyanide p-triﬂuoromethoxyphenylhydrazone (FCCP) and 6 mM Rotenone and Antimycin A (Rot/AA) were added to determine (B) baseline, (C) maximal, and (D) complex 2 OCR. (E) Maximal respiration minus baseline respiration was used to calculate SRC (n=9 wells per group). All bar graphs are expressed as mean6SD. *P,0.05, IDH2 versus control; **P,0.05, IDH2 versus mock; #P,0.05, myc Idh2 versus control; $P,0.05, myc Idh2 to mock.

J Am Soc Nephrol 29: 1154–1164, 2018 IDH2 Prevents AKI 1157 BASIC RESEARCH www.jasn.org

Figure 4. IDH2 localizes to the mitochondria and alters function in vivo. (A) Representative immune blot of protein (5 mg) from mitochondria extracts. Lanes 1–3 are saline only, and lanes 4–6 are myc-tagged IDH2 (left panel). Band densitometry (right panel) of ratio of myc-tagged IDH2/Actin is shown (n=4 per group). *P,0.05, myc IDH2 versus vehicle. (B–F) TMRM-labeled mitochondria by intravital microscopy in (B) sham rats, (C) rats after injection of saline, (D) Idh2, or (E) IPC. (F) Intensities from each group were then averaged, giving a mean TMRM intensity value (n=50 tubules per rat). DT, distal tubule; PT, proximal tubule. Scale bar, 20 mm. *P,0.05, IDH2 versus vehicle; #P,0.05, IPC versus vehicle. (G) Respiration of isolated renal mitochondria 1 week after saline or Idh2 injections. Oxygen ﬂux was assayed after addition of isocitrate, malate, pyruvate, succinate, and ADP (n=3 per group). All bar graphs are expressed as mean6SD. IMD, isocitrate, malate, and ADP; IMPD, isocitrate, malate, pyruvate, and ADP; IMPSD, isocitrate, malate, pyruvate, succinate, and ADP. *P,0.05, Idh2 versus vehicle; #P,0.05, Idh2 versus vehicle; $P,0.05, Idh2 versus vehicle.

Mitochondria polarization was evaluated with TMRM and in- serum creatinine (Figure 5A) and improved tissue histology travital imaging. After 2 weeks of IPC (i.e.,14daysafterrecovery in response to IRI (Figure 5, B–E). Analysis of mitochondria from 35 minutes of IRI), the TMRM signal from rat kidney 60 minutes after reperfusion showed that IDH2 preserved re- tubules was significantly elevated relative to that observed in spiratory capacity relative to vehicle controls (Figure 5, F and sham IPC control rats (Figure 4, B and E). Similarly, rats sub- G). Finally, IPC and Idh2 delivery both increase ATP levels jected to Idh2 delivery have significantly increased TMRM signal versus sham and saline-injected control animals, indicating relative to saline-injected control animals (Figure 4, C, D, and F). that IDH2 enhances ATP stores, thereby protecting against To evaluate whether Idh2 delivery in vivo influenced mitochon- IRI (Figure 5H). dria respiration, renal cortical mitochondria were isolated and analyzed using oximetry. Respiration was measured in response to isocitrate, pyruvate, and succinate to evaluate com- DISCUSSION plexes 1 and 2 activity. Mitochondria isolated from Idh2-treated animals showed increased state 2 and state 3 respiration AKI remains a significant cause of comorbidity in the hospital relative to saline-injected animals, regardless of the substrate setting, with supportive care as the sole treatment available. used (Figure 4G). Therefore, measures that prevent AKI are likely to garner rapid clinical acceptance particularly, because AKI is associated with IDH2 Increases Oxidative Phosphorylation and increased mortality and risk of progression to ESRD.10,23–26 Prevents Tissue Injury Among the potential clinical interventions available to prevent To determine if Idh2 delivery mimics IPC, we subjected rats to AKI, IPC has been shown to activate cellular adaptations that either IPC or Idh2 delivery and allowed them to recover. As confer resistance to subsequent ischemic stress.8,27,28 How- expected, IPC-treated rats were resistant to IRI as indicated by ever, clinical trials of IPC have failed to show consistent pro- reduced serum creatinine 24 hours after IRI relative to sham IPC. Interestingly, Idh2 resulted in a similar reduction in tection against AKI, and the variance between tested protocols

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Figure 5. Idh2 mimics IPC. (A) Comparison of serum creatinine (sCr) after IPC or Idh2 delivery 24 hours after IRI (n=5 per group). *P,0.05, Idh2 versus vehicle; #P,0.05, IPC versus vehicle. (B–D) Hematoxylin and eosin–stained section of renal outer medulla after IRI in rats treated with (B) saline, (C) Idh2 injection, or (D) IPC is shown, and corresponding injury scoring is in E. Scale bar, 20 mm. *P,0.05, IRI IDH2 versus IRI saline; #P,0.05, IRI IPC versus IRI saline. Mitochondria respiration 1 hour after IRI in vehicle- or Idh2-treated rats is shown with (F) pyruvate or (G) succinate as a substrate (n=7 per group). *P,0.05, Idh2 IRI versus vehicle IRI. (H) Renal ATP-to-AMP ratios are shown in sham-, vehicle-, Idh2-, and IPC-treated rats (n=3 per group). All bar graphs are expressed as mean6SD. *P,0.05, Idh2 versus vehicle; #P,0.05, IPC versus sham.

make it impossible to identify the root cause for success or maintains mitochondria functionality rather than biochemi- failure. cal purity. Many of the proteins in the proteome of precondi- Mitochondria are important regulators of normal physiol- tioned mitochondria were readily identified as belonging to ogy. Recently, loss of genes, such as APOL-1, has pointed to the oxidation phosphorylation, fatty acid metabolism, and Reiske importance of mitochondria in regulating disease, specifically protein pathways (Table 1). The cell signaling systems respon- kidney diseases.29,30 AKI targets renal mitochondria and ini- sible for these changes in mitochondria composition are still tiates bioenergetic changes that alter the relative contribution unclear. Preliminary work by our laboratory failed to identify of oxidative phosphorylation and glycolysis to cellular ATP any changes in endogenous metabolites observed by Hato stores.11,31 Substantial bodies of evidence point to adaptive et al.41 in response to endotoxin, and we were not able to changes in mitochondria protein composition mediated by observe an increase in ROS that may account for altered mi- PGC-1a and TFAM after IPC.32–35 Changes in cellular local- tochondria protein gene transcription.42 Our findings suggest ization of GSK3-b and hexokinase fundamentally alter the that more work is needed to identify the adaptive signaling in propensity of the mitochondria transition pore to open, response to IPC. thereby inducing a cell death cascade.36,37 Additional changes To test whether a protein identified in the proteomic screen in Cm and state 1/3 mitochondria respiration have also been was of potential use for conferring resistance to ischemia, we reported in response to IPC.38–40 Taken together, interven- used a cell culture hypoxia injury system to assess a candidate’s tions designed to alter mitochondria physiology in a manner efficacy in conferring protection against hypoxia. The MPTC similar to IPC are also likely to confer cellular resistance to line used in this study has been grown under conditions, ischemic injury in a reliable way. whereby virtually all of their intracellular ATP is produced In this communication, we use a proteomic analysis of mi- by oxidative phosphorylation.43 Because the cells require tochondria isolated from the renal cortex of ischemic precon- 38% oxygen concentration to maintain growth, these cells ditioned Sprague–Dawley rats using a timed renal pedicle are susceptible to hypoxic injury.44 These qualities make the crossclamp to induce the preconditioned state. Mitochondria cell line uniquely appropriate for use in hypoxia injury studies were isolated 2 weeks after IPC using an isolation method that to screen candidate genes to determine if they confer

J Am Soc Nephrol 29: 1154–1164, 2018 IDH2 Prevents AKI 1159 BASIC RESEARCH www.jasn.org resistance to hypoxic injury. In our assays for cell injury, using (Figure 1B). This is seen in reductions of serum creatinine and an assay that predominantly measures cell necrosis, S3 cells improved tissue morphology in our animal model of kidney transfected with either N-terminal myc-tagged Idh2 or Idh2 injury (Figures 1G and 5A). We also found that both IPC and were resistant to hypoxic injury. In vitro transfection models Idh2 delivery increase states 1–3 respiratory capacity. This re- indicate that IDH2 localized to the mitochondria (Figure 1, C– sult stands in contrast to that of the report by Han et al.,59 in G). IDH2 serves both reductive and oxidative functions rang- which IPC decreased IDH2 protein levels; however, our im- ing from glutathione reduction to NAD(P)H generation de- mune blots were performed on protein lysates made from pending on cellular requirements.12,45–49 To study these kidney cortex, whereas Han et al.59 used whole-kidney lysates. changes, we analyzed membrane potential and respiration in Their work concluded that downregulation of IDH2 expres- both in vivo and in vitro models. Our data indicate that mito- sion resulted in increased injury after AKI.59 Taken together, chondria membrane potential was increased in Idh2 transfec- Idh2 delivery mimics IPC, particularly in the ability to confer ted cells compared with controls. Changes in mitochondria protection against subsequent ischemic injury. Because it has membrane potential are linked with changes in reduction po- been difficult to reproduce the protective effect of IPC in the tential (i.e., electron movement), leading to increases in oxi- clinical setting, it is possible that timely gene delivery offers an dative phosphorylation. Our data indicate that IPC or retro- alternative method to prevent ischemic injury. We believe that grade injection of Idh2 protects mitochondria from IRI and IDH2 gene delivery will mimic IPC, because it has been pre- leads to increased reduction potential compared with our con- viously reported that its loss is implicated with promoting trol animals (Figure 4, B–F), and these increases are linked to ischemic disease.59 cell survival and avoidance of cell death pathways.50 It is our hypothesis as well as that of many others that, after injury, mitochondria revert to a Warburg state manifest by a shift CONCISE METHODS to glycolysis.51–53 This allows accumulation of glycolytic metabolites, lipids, and nucleic acids that will aid in cellular Materials repair.51–53 However, increases in mitochondria membrane po- All fluorescent probes were purchased from Thermo Fisher Scientific tential in response to preconditioning or Idh2 retrograde in- (Waltham, MA). IDH2 antibody was purchased from Protein Tech jection indicate that this glycolytic shift may not occur. Idh2 (Chicago, IL). The c-MYC antibody, (9e10) developed by J.M. Bishop acts to increase metabolic output in preparation for injury. at the University of California, San Francisco was obtained from the Strikingly, our analysis of mitochondria respiration found Developmental Studies Hybridoma Bank created by the National In- increased states 1 and 2 respiratory capacity in Idh2 trans- stitute of Child Health and Human Development of the National fected cells. Our results indicate that gene transfection leads Institutes of Health and maintained at The University of Iowa, De- to increases in baseline, maximal, and complex 2 respiration partment of Biology in Iowa City, Iowa. b-Actin was purchased from (Figure 3), indicative of increased oxidative phosphorylation. Millipore (Billerica, MA). All secondary antibodies were purchased Interestingly, Idh2 transfection also increased SRC, which has from Jackson Laboratories (West Grove, PA). All plasmid DNA was been linked to cell survival of cells prone to oxidative purchased from Origene (Rockville, MD). FIJI was downloaded from stress.54–57 Idh2 transfection increases SRC, indicating that, the National Institutes of Health website (National Institutes of before injury onset, cells are primed to handle oxidative stress Health, Bethesda, MD).60 Lipofectamine 3000 was purchased from and allocate the additional energy to aid in cellular recovery. Thermo Fisher Scientific. Seahorse reagents and kits were purchased The increase in SRC is linked with decreases in serum creat- from Agilent (Santa Clara, CA). inine, and reductions in hypoxia induced cell death. Data from cell culture studies suggest a fundamental shift in mi- Animals and Animal Care and Use tochondria physiology, in which additional Idh2 expression Male Sprague–Dawley rats (250–350 g; Envigo, Indianapolis, IN) resulting from exogenous gene delivery increases metabolic were used for our in vivo studies. Rats had access to food and water, flux through the mitochondria aided by an increase in mem- and all experiments conducted followed National Institutes of Health brane potential. To further confirm these findings, we next guidelines. Rats were randomly assigned to control or experimental examined whether exogenous gene delivery in vivo would groups. Approval from the Indiana University School of Medicine recapitulate cytoprotection in vitro. Institutional Animal Care and Use Committee and the Richard L. We recently reported successful gene transfer to kidneys Roudebush Veterans Affairs Medical Center Animal Care and Use using hydrodynamic gene delivery.19 An important finding Committee was gained before all in vivo studies. in this work suggested that it is possible to use a mammalian expression vector to alter renal physiology on an organ-wide Cell Lines scale.58 Our data in this communication support this conjec- S3 proximal tubule cells were grown as previously described.61 Val- ture. Immune blots confirm our proteomic findings that idation of the cell line origin was performed by IDEXX BioResearch IDH2 levels increase in IPC, suggesting a possible role in pre- (Columbia, MO), and the cells have the following STR profile: MCA- vention of AKI. Similarly, retrograde injections of Idh2 lead to 4–2: 20.3; MCA-5–5: 17, 18; MCA-6–4: 14; MCA-6–7: 12, MCA-9–2: equivalent increases in IDH2 and protection against ischemia

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15; MCA-12–1: 16, 17; MCA-15–3: 22.3; MCA-18–3: 17; MCA-X-1: 7% FBS, and 4.1% sodium bicarbonate solution (Sigma-Aldrich Corpo-

28. ration, St. Louis, MO) and grown at 37°C, 5% CO, and 38% O2. National Institutes of Health 3T3 cells62 were purchased from National Institutes of Health 3T3 cells62 were cultured in DMEM es- American Type Culture Collection (Manassas, VA) and maintained sential media (American Type Culture Collection) supplemented with 10% in culture according to American Type Culture Collection recom- FBS and 1% penicillin-streptomycin and grown at 37°C in a 5% CO2 mendations. incubator.

IPC, Gene Delivery, and AKI Induction Immunohistochemical Staining and Colocalization Bilateral renal IRI (AKI induction) was induced by 35-minute renal National Institutes of Health 3T3 cells were transfected with Lipofect- pedicle clamping in animals anesthetized with aketamine (100 mg/kg) amine 3000 with myc-tagged Idh2 cDNA or mock transfected accord- plus xylazine (10 mg/kg) plus acepromazine (2.5 mg/kg) cocktail as ing to the manufacturer’sdirections(ThermoFisherScientific). described previously.58 IPC was performed by allowing animals to Immunohistochemical staining was conducted on myc-tagged Idh2 recover from a 30-minute bilateral renal pedicle clamp for 14 days.63 cDNA and mock transfected NIH 3T3 cells using anti–c-myc (9e10) IPC efficacy was verified by a second bout of IRI. Idh2 gene delivery antibody and FITC donkey anti-rabbit as previously described.68 The cells was performed as previously described.19 For analysis of renal func- were also stained with Hoechst 33342 and 100 nM Mitotracker red. Cells tion, serum creatinine was measured as previously described.58 were imaged using an Olympus FV 1000 confocal microscope. Blue, red, and green channels were used to analyze colocalization, and each channel Proteomics was collected in sequential mode to avoid crosstalk. Manders colocaliza- Mitochondria were isolated from IPC and sham IPC renal cortex, and tion tests were conducted as described by Dunn et al.22 they were stored at 280°C and used for proteomics as previously described.64,65 Isolation and Analyses of Mitochondria Kidney cortex was homogenized in mitochondria isolation buffer

Intravital Imaging to Mitochondria Using TMRM (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, Mitochondria structure and function were evaluated using TMRM and 1.0 mM EDTA, pH 7.9) using a PBI S3 shredder (Pressure Bio- according to the methods described by Hall et al.66 sciences, Easton, MA). The homogenate was centrifuged at 8003g for 10 minutes at 4°C. The supernatant was collected and centrifuged at JC-1 Cell Polarization 14,0003g for 10 minutes. Mitochondria pellet was suspended in mi- MPTCs were transfected using Lipofectamine 3000 according to the tochondria isolation buffer and then centrifuged at 14,0003g for 10 manufacturer’s specifications (Thermo Fisher Scientific). Two days minutes. The pellet was suspended in mitochondria isolation buffer after transfection, cells were plated into a 96-well plate. Twenty-four and used immediately for respiration analysis. Mitochondria protein hours later, the JC-1 assay was performed as follows. JC-1 (5 mM) was content was determined using a Bradford assay (Bio-Tek Instruments added to each well. Media and cells were placed in a hypoxia chamber Inc., Winooski, VT). Mitochondria respiration was measured using for 30 minutes. After hypoxia, fluorescence was measured using a an Oroboros Oxygraph-O2k (Oroboros Instruments, Innsbruck, Molecular Devices SpectraMax M5 microplate reader (Molecular De- Austria) as previously described.69,70 Equivalent amounts of mito- vices, Sunnyvale, CA) with an excitation wavelength of 488 nm. Emis- chondria protein were loaded into a final volume of 2.0 ml miRO5 sion was measured at both 527 and 590 nm. (110 nm sucrose, 20 mM HEPES, 20 mM taurine, 60 mM K-lacto- 21 bionate, 3 mM MgCl2,10mMKH2PO4, 0.5 mM EGTA, and 1 g L Analyses of Nucleotides by HPLC BSA, pH 7.1) respiration buffer. After addition of isolated mitochon- Animals were anesthetized using thiobutabarbital (Inactin), and kid- dria, oxygen flux was allowed to stabilize before addition of substrates neys were exteriorized, and frozen in situ with liquid nitrogen–cooled or ADP. Substrates were added in the following order and diluted to sheet metal pliers. For analysis of ATP, cell extracts were isolated and the following concentrations: 10 mM Isocitrate, 2.5 mM Malate, and prepared for HPLC as previously described.67 20 nM ADP; 7.5 mM Pyruvate and 20 nM ADP; and 10 mM Succinate, Cell extracts were isolated from MPTCs and prepared for HPLC as and 20 nM ADP. Data were analyzed using Oroboros Datlab version 6 previously described.67 (Oroboros Instruments), and calibration before experimentation was conducted according to Oroboros protocols. Immune Blotting Immune blotting was performed as previously described.68 The fol- Analyses of Cellular Respiration Using a Seahorse XF lowing antibodies were used for analysis: IDH2 (NADP+), myc 96 Analyzer (9e10), and b-actin. FIJI immune blot module was used to analyze Mitochondria respiration was conducted using a Seahorse XF 96 extra- band intensities.60 cellular flux analyzer as previously described71; 5000 MPTCs were plated in a Seahorse XF 96 cell culture microplate 24 hours before analysis. Murine Cell Culture Analysis was performed with the appropriate growth media containing MPTCs were cultured in modified essential media (50:50 HAM F-12: 10 mM glucose, 100 mM sodium pyruvate, and 1 M glutamine. Cells were DMEM with L-glutamine, HEPES, sodium selenite, sodium pyruvate, allowed to acclimate for 1 hour before analysis. Baseline measurements phenol red, and insulin) supplemented with 1% penicillin-streptomycin, were conducted for transfected and mock transfected cells before

J Am Soc Nephrol 29: 1154–1164, 2018 IDH2 Prevents AKI 1161 BASIC RESEARCH www.jasn.org compound addition. Respiration measurements were conducted after B.A.M. is a founder of Fast Diagnostics and a paid consultant to Mitobridge. injections of 7 mMcarbonylcyanidep-trifluoromethoxyphenylhydrazone R.L.B. is a founder of Rene Medical. and 6 mM Rotenone/Antimycin A. All flux data are expressed in OCR in picomoles per minute per cell. REFERENCES Cell Transfections In total, 2.03105 cells were plated onto a six-well plate the day before 1. Murry CE, Jennings RB, Reimer KA: Preconditioning with ischemia: A delay of lethal cell injury in ischemic myocardium. Circulation 74: 1124– transfection. Twenty-four hours later, cells were transfected with ei- 1136, 1986 ther Idh2 cDNA or myc-tagged Idh2 cDNA using Lipofectamine 3000 2. Basile DP, Yoder MC: Renal endothelial dysfunction in acute kidney according to the manufacturer’s specifications. All subsequent studies ischemia reperfusion injury. 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Basile DP, Anderson MD, Sutton TA: Pathophysiology of acute kidney 22 alization was determined using the Manders localization analysis. All bar injury. Compr Physiol 2: 1303–1353, 2012 graphs are expressed as mean6SD. All experiments were conducted in 11. Jassem W, Fuggle SV, Rela M, Koo DD, Heaton ND: The role of mito- duplicate with at least one control group and one experimental group to chondria in ischemia/reperfusion injury. Transplantation 73: 493–499, 2002 ensure random assortments. We were blinded for only injury scoring. 12. Hurley JH, Dean AM, Koshland DE Jr, Stroud RM: Catalytic mechanism of NADP(+)-dependent isocitrate dehydrogenase: Implications from the structures of magnesium-isocitrate and NADP+ complexes. Biochemistry 30: 8671–8678, 1991 ACKNOWLEDGMENTS 13. Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, Kos I, Batinic-Haberle I, Jones S, Riggins GJ, Friedman H, Friedman A, Reardon D, Herndon J, Kinzler KW, Velculescu VE, Vogelstein B, Bigner DD: IDH1 The authors thank the staff of the Indiana Center for Biological and IDH2 mutations in gliomas. NEnglJMed360: 765–773, 2009 Microscopy for their expert advice and support. The authors also 14. Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, Mankoo thank Dr. Robert Harris for many helpful discussions. P, Carter H, Siu IM, Gallia GL, Olivi A, McLendon R, Rasheed BA, Keir S, J.A.C. is supported by National Institutes of Health grant T32 Nikolskaya T, Nikolsky Y, Busam DA, Tekleab H, Diaz LA Jr, Hartigan J, Smith DR, Strausberg RL, Marie SK, Shinjo SM, Yan H, Riggins GJ, HL07995. B.A.M. is supported by the O’Brien Center for Advanced Renal Bigner DD, Karchin R, Papadopoulos N, Parmigiani G, Vogelstein B, Microscopic Analysis at the Indiana Center for Biological Microscopy. B. Velculescu VE, Kinzler KW: An integrated genomic analysis of human A.M. received grant support from National Institutes of Health grants glioblastoma multiforme. Science 321: 1807–1812, 2008 DK091623 and DK079312 and the Veterans Administration through a 15. Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, Merit Review award. D.P.B. is supported by National Institutes of Health Fantin VR, Jang HG, Jin S, Keenan MC, Marks KM, Prins RM, Ward PS, grant DK063114. R.L.B. is supported by Veterans Administration Merit Yen KE, Liau LM, Rabinowitz JD, Cantley LC, Thompson CB, Vander Heiden MG, Su SM: Cancer-associated IDH1 mutations produce 2- award BX001736. S.A.J. was awarded grants from the National Institutes hydroxyglutarate. 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1164 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1154–1164, 2018 SIGNIFICANCE STATEMENT

Ischemic preconditioning is a physiologic adaptation to a transient ischemic event that confers protection against subsequent ischemic injury. Depending on the organ under study and the method of ischemic preconditioning, the adaptive response to ischemia is variable. Although some of the signaling pathways responsible for ischemic preconditioning have been identiﬁed, the mechanism of cellular adaptation remains incompletely understood. In this communication, we show that delivering plasmids encoding isocitrate dehydrogenase 2 to kidneys confers resistance to subsequent ischemic injury and increases states 2 and 3 mitochondria respiration. These are similar changes to those observed in mitochondria isolated from ischemic preconditioned kidneys. This work shows that it is possible to use gene delivery to confer resistance to subsequent ischemic injury.