Dynamin-Related Protein 1 Deficiency Promotes Recovery From

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Dynamin-Related Protein 1 Deﬁciency Promotes Recovery from AKI

Heather M. Perry ,1 Liping Huang,1 Rebecca J. Wilson,2 Amandeep Bajwa,1 Hiromi Sesaki,3 Zhen Yan,2 Diane L. Rosin,4 David F. Kashatus,5 and Mark D. Okusa1

1Department of Medicine, Division of Nephrology and Center for Immunity, Inﬂammation, and Regenerative Medicine, 2Department of Medicine, Division of Cardiovascular Medicine and Center for Skeletal Muscle Research at the Robert M. Berne Cardiovascular Research Center, and 4Departments of Pharmacology and 5Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, Virginia; and 3Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland

ABSTRACT The proximal tubule epithelium relies on mitochondrial function for energy, rendering the kidney highly susceptible to ischemic AKI. Dynamin-related protein 1 (DRP1), a mediator of mitochondrial fission, regulates mitochondrial function; however, the cell-specific and temporal role of DRP1 in AKI in vivo is unknown. Using genetic murine models, we found that proximal tubule–specific deletion of Drp1 prevented the renal ischemia-reperfusion–induced kidney injury, inflammation, and programmed cell death observed in wild-type mice and promoted epithelial recovery, which associated with activation of the renoprotective b-hydroxybutyrate signaling pathway. Loss of DRP1 preserved mitochondrial structure and reduced oxidative stress in injured kidneys. Lastly, proximal tubule deletion of DRP1 after ischemia-reperfusion injury attenuated progressive kidney injury and fibrosis. These results implicate DRP1 and mitochondrial dynamics as an important mediator of AKI and progression to fibrosis and suggest that DRP1 may serve as a therapeutic target for AKI.

J Am Soc Nephrol 29: 194–206, 2018. doi: https://doi.org/10.1681/ASN.2017060659

In patients with AKI who survive, there is an in- important contributor to CKD.5 Mitochondria are creased risk of developing CKD and ESRD.1 A recent dynamic organelles that have crucial roles in energy systematic review and meta-analysis provided production, metabolism, calcium homeostasis, and strong evidence that identiﬁed AKI as an indepen- programmed cell death. The proximal tubule is rich dent risk factor for CKD, ESRD, and death.2 The in mitochondria and highly dependent on oxidative mechanisms underlying the AKI to CKD contin- phosphorylation for the large demand of ATP nec- uum are not well understood but have recently essary for cellular function, notably solute trans- been reviewed.1,3 port.5,6 During renal ischemia, mitochondrial Mitochondrial dysfunction is important in the function diminishes,7 and within 15 minutes of pathogenesis of AKI4 and has also emerged as an

Significance statement Received June 19, 2017. Accepted September 29, 2017. AKI can lead to CKD. Mitochondrial dysfunction in the Published online ahead of print. Publication date available at proximal tubule epithelium has emerged as an impor- www.jasn.org. tant factor in this progression. Mitochondria are dy- Correspondence: Dr. Mark D. Okusa, Department of Medicine, namic organelles, and their shape is closely linked to Division of Nephrology and Center for Immunity, Inflammation, function. This study provides evidence that proximal and Regenerative Medicine, University of Virginia, PO Box tubule deletion of Drp1, a mediator of mitochondrial 800133, Charlottesville, VA 22908. Email: [email protected] fission, prevents AKI and may have therapeutic value as a target after injury to prevent progressive fibrosis. Copyright © 2018 by the American Society of Nephrology

194 ISSN : 1046-6673/2901-194 J Am Soc Nephrol 29: 194–206, 2018 www.jasn.org BASIC RESEARCH reperfusion, ultrastructural analysis reveals fragmented mito- epithelium highly susceptible to IRI.17 A direct reporter of chondria.8 Over time, there is organelle swelling and disrup- mitochondrial health and oxidative stress in the outer medulla tion of the tightly packed cristae.9 These morphologic changes in vivo has not been shown. To address this, we crossed the coincide with mitochondrial dysfunction and ATP depletion CAG-CAT-MitoTimer mice (CMV enhancer-b-actin pro- and precipitate cell death pathways. Mitochondrial-derived moter driving LoxP-flanked chloramphenicol acetyltransfer- reactive oxygen species (ROS) can also contribute to oxidative asegenewithastopcodonbeforetheMitoTimer reporter stress during AKI. Emerging evidence has shown that main- gene)18 with the proximal tubule–specific PepCKCre+ taining mitochondrial structural integrity10 and boosting mi- mouse19 to generate a PepCKCre MitoTimer mouse. The Mito- tochondrial biogenesis11–14 may promote kidney recovery Timer reporter gene has a mitochondrial targeting sequence from AKI and prevent fibrosis. Much less is understood about fused to the Timer fluorescence-based reporter gene,18 which mitochondrial remodeling in progression to CKD. is a modified DsRed mutant that fluoresces green when newly Mitochondrial morphology and function are tightly coor- synthesized and irreversibly shifts to red after oxidation (de- dinated. Mitochondrial morphology is dynamic and can rap- hydrogenization) of the Tyr-67 residue. In PepCKCre Mito- idly respond to metabolic and cellular stress responses. The Timer kidneys, MitoTimer fluorescence was only detected in relative balance of fission and fusion dictates mitochondrial the CD13+ proximal tubule epithelium of the outer medulla shape and can result in a spectrum from fragmented to a hyper- (Figure 1A). To determine if proximal tubule mitochondria fused network. Fusion is associated with increased ATP pro- in the outer medulla become oxidized after IRI, kidneys of duction, whereas fission is associated with impaired oxidative PepCKCre MitoTimer mice were subjected to 26 minutes of phosphorylation and ROS generation. Fragmentation occurs during normal physiologic processes to facilitate mitochondrial transport, clearance of damaged mitochondria, and apoptosis, but it can also result from cellular dysfunction. Fission sites are marked by contacts with the endoplasmic reticulum, where the fission machinery is recruited, including the GTPase dynamin- related protein 1 (DRP1). DRP1 dimers and oligomers assemble into a helical structure, and constriction of the helix promotes severing of the mitochondrion.15 DRP1 is activated in kidney tubule epithelial cells during conditions of ATP depletion in vitro. In response to ATP depletion, DRP1 is dephosphorylated on S63716 and accumulates on mitochondria.8 ATP depletion or exposure to cisplatin, a neph- rotoxic agent, results in mitochondrial fragmentation and apoptosis, which can be abrogated by expression of a dominant negative DRP1 or Drp1 (also known as Dnm1l) knockdown. Pretreatment of mice with the putative pharmacologic inhibitor of DRP1, mdivi-1, results in protection against ischemia-reperfusion injury (IRI).8 On the basis of these data, we hypothesized that genetic deletion of Drp1 in the proximal tubule epithelium protects against IRI. Furthermore, we tested the therapeutic potential of Drp1 deletion in the proximal tubule epithelium with a genetically inducible mouse. We showed that genetic deletion of Drp1 specifically in the proximal tubule epithelium before renal IRI protects against kidney injury. Additionally, delayed deletion of proximal tubule Drp1 in the recovery period after IRI resulted in improved kidney recovery and reduced fibrosis. These results Figure 1. Outer medulla mitochondria become oxidized after IRI. indicate that proximal tubule DRP1 increases kidney suscepti- Kidneys of PepCKCre MitoTimer mice were subjected to 26 min- bility to IRI and contributes to the progression of AKI to fibrosis. utes of unilateral ischemia and 3 or 6 hours of reperfusion. (A) Representative images of native MitoTimer fluorescence of indi- vidual green (left panel; unoxidized) and red (center panel; oxidized) RESULTS channels and MitoTimer merged with CD13+ proximal tubule immunofluorescence (right panel) in control kidneys. (B) Representative images and (C) quantification of MitoTimer in the unoxidized and MitoTimer Mouse Reveals IRI-Induced Mitochondrial oxidized state in mitochondria in proximal tubule cells in the medulla. Oxidative Stress in the Outer Medulla IRI indicates the kidney exposed to unilateral ischemia-reperfusion The already hypoxic countercurrent exchange properties of the (operated kidney). Con, contralateral control (unoperated kidney). kidney vasa recta render the outer medulla proximal tubule Scale bar, 10 mm. *P,0.05; ***P,0.001.

J Am Soc Nephrol 29: 194–206, 2018 Proximal Tubule DRP1 in AKI 195 BASIC RESEARCH www.jasn.org unilateral ischemia and 3 or 6 hours of reperfusion. The red- Mff, Mfn1, and Opa1 were similar across genotype and surgery to-green ratio of signal intensity significantly increases at 3 groups. Mfn2 levels were decreased after IRI in Drp1WT kidneys and 6 hours of reperfusion compared with the contralateral and similar to sham levels in Drp1PTKO IRI kidneys (Supple- control kidneys (Figure 1, B and C). Data show that IRI in- mental Figure 1A). An increase (twofold of baseline) in mito- duced mitochondrial oxidative stress in the proximal tubule chondrial mass was observed in Drp1WT kidneys at 6 hours of epithelium in the outer medulla. reperfusion, with return to baseline levels at 24 hours, but this increase was not found in Drp1PTKO kidneys. Mitochondrial Deletion of Proximal Tubule Drp1 Protects Kidneys mass in Drp1PTKO kidneys did not change at 6 and 24 hours of from IRI reperfusion relative to baseline (at 0 hours) (Supplemental Fig- Drp1 can be activated and mediates mitochondrial fission in ure 1B). IRI-induced increases in plasma creatinine (PCr) lev- response to oxidative stress and mitochondrial dysfunction.8 els, acute tubular necrosis (ATN), and kidney injury markers, The cell-specific role of DRP1 in AKI has not been determined kidney injury molecule 1 (Kim1) and neutrophil gelatinase– in vivo. To test the hypothesis that DRP1 in the proximal tu- associated lipocalin, in Drp1WT mice at 24 hours of reperfusion bule epithelium promotes IRI, proximal tubule–specific Drp1 were attenuated in Drp1PTKO mice (Figure 2, B–E, Supplemen- fl fl knockout mice PepCKCre+ Drp1 / (hereafter referred to as tal Figure 2). At 6 hours of reperfusion, ATN was reduced in fl fl Drp1PTKO) and littermate control PepCKCre2 Drp1 / (Drp1WT) Drp1PTKO compared with Drp1WT kidneys (Figure 2C), and mice were subjected to 26 minutes of bilateral renal is- PCr (Figure 2B) and injury markers were not significantly dif- chemia or sham surgery, and kidney function, tissue injury, ferent (Figure 2, D and E). and transcript levels were measured after 6 and 24 hours of Inflammation is a key characteristic of AKI.20,21 IRI- reperfusion. Drp1 transcript levels were reduced in Drp1PTKO induced increases in transcript levels of proinflammatory cyto- whole-kidney tissue lysate in sham-operated mice and at both kines chemokine (C-C motif) ligand 2 (Ccl2)andIl6 were 6 and 24 hours of reperfusion compared with Drp1WT levels similar at 6 hours of reperfusion in Drp1WT and Drp1PTKO (Figure 2A). Transcript levels of fission and fusion mediators kidneys (Figure 2, F and G). Additionally, the numbers of

Figure 2. Proximal tubule–specificdeletionofDrp1 abrogates IRI. PepCKCre- Drp1fl/fl (Drp1WT)andlittermatePepCKCre+ Drp1fl/fl (Drp1PTKO) mice were randomized to bilateral 26 minutes of ischemia or sham operation and euthanized after 6 and 24 hours of reperfusion. (A) Kidney Drp1 relative to Gapdh transcript levels and (B) PCr levels. (C) Stereologic quantification of ATN in the outer medulla expressed as a percentage of total surface area of kidney outer medulla. Kidney (D) Kim1 and (E) neutrophil gelatinase– associated lipocalin (Ngal) relative to Gapdh transcript levels. Kidney (F) Ccl2 and (G) Il6 relative to Gapdh transcript levels. (H) Quantitation of renal neutrophils (CD11b+Ly6G+)byflow cytometry. *P,0.05; **P,0.01; ***P,0.001; ****P,0.001.

196 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 194–206, 2018 www.jasn.org BASIC RESEARCH

Figure 3. Loss of Drp1 in proximal tubules preserves mitochondrial structure, enhances mitochondrial function, and reduces oxidative stress. Drp1WT and littermate Drp1PTKO mice were subjected to bilateral IRI and euthanized after (A and B) 15 minutes or (C–F) 6 and 24 hours of reperfusion. (A) Electron micrographs and (B) quantiﬁcation of mitochondrial contour measurements. Data are means6SEM of mitochondrial contour measurements from six to eight proximal tubules per mouse from n=3 mice per group. Arrowheads indicate the edge of the proximal tubule on the basolateral side. n, Nucleus. Scale bar, 1 mm. Kidney (C) Nrf2 and (D) Ho1 relative to Gapdh transcript levels and (E) NRF2 protein (100 kD) and b-tubulin (bTub; 55 kD) levels by Western blot from two independent experiments. (F) Oxygen consumption rate (OCR) and quantiﬁcation of metabolic parameters by Seahorse in primary tubule epithelial cells isolated

J Am Soc Nephrol 29: 194–206, 2018 Proximal Tubule DRP1 in AKI 197 BASIC RESEARCH www.jasn.org kidney neutrophils at 6 hours were not different (25.7568.49 Table 1. Contour measurements of mitochondria from versus 19.8368.04 cells per 1 high-powered field, respec- ultrastructural analysis tively). Il6 levels and neutrophil numbers were attenuated in Control IRI PTKO Parameter Drp1 kidneys at 24 hours of reperfusion (Figure 2, G and Drp1WT Drp1PTKO Drp1WT Drp1PTKO H). Altogether, our data provide evidence that DRP1 promotes 6 6 6 a 6 fl Roundness 0.478 0.019 0.489 0.019 0.554 0.016 0.485 0.025 kidney injury and in ammation at 24 hours of reperfusion Aspect 0.67460.015 0.68360.015 0.73460.012a 0.68260.019 after IRI. ratio Shape 4.4360.080 4.3860.070 4.15260.050 4.51760.110b Loss of DRP1 Reduces Mitochondrial Dysfunction and factor Promotes Epithelial Cell Recovery aP,0.05 compared with control Drp1WT. b , WT DRP1 executes mitochondrial fission in response to cellular P 0.05 compared with IRI Drp1 . signals, and IRI-induced mitochondrial fragmentation can occur within 15 minutes of reperfusion. To determine mito- the loss of DRP1 enhances mitochondrial function. Consis- chondrial morphology in vivo, kidneys were collected for ul- tent with increased mitochondrial function in primary epithe- trastructural analysis by electron microscopy after ischemia lial cells from kidneys of Drp1PTKO mice, there was a decrease and 15 minutes of reperfusion or no surgery, and a quantita- in the percentage of cells with mitochondria in a fragmented tive assessment of ultrastructural contour analysis was per- state compared with control cells (Figure 3G). formed on cortical proximal tubule mitochondria. Mitochondria Mitochondrial dysfunction can contribute to programmed from Drp1WT mice were shorter and rounder after ischemia cell death. The number of terminal deoxynucleotidyl transferase– (Figure 3, A and B, Table 1) compared with no surgery. Mito- mediated digoxigenin-deoxyuridine nick-end labeling– chondria in Drp1PTKO IRI kidneys were larger but similar in positive (TUNEL+) cells was similar at 6 hours of reperfusion shape compared with no surgery and larger, longer, and less and reduced at 24 hours of reperfusion in IRI kidneys of Drp1PTKO circular compared with Drp1WT IRI kidneys (Figure 3, A and mice compared with control mice (Figure 4). No TUNEL+ B, Table 1), suggesting that Drp1 deletion in the proximal tubule cells were observed in sham animals (data not shown). Given preserves mitochondrial morphology and prevents IRI-induced these observations (similar TUNEL and injury markers fragmentation. reduced at 24 hours), we hypothesized that Drp1 null tubules Mitochondrial dysfunction occurs during IRI and contrib- may be able to proliferate and recover by 24 hours of reperfusion. utes to oxidative stress. Oxidative stress can induce nuclear As shown in Figure 3, Ki67+ cells were detected in IRI kidneys of factor, erythroid derived 2, like 2 (NRF2), a transcription factor Drp1PTKO mice, but very few, if any, were detected in IRI kidneys that regulates expression of cellular redox balance, mitochon- from Drp1WT mice. These data provide evidence that deletion of drial proteins, and cytoprotective factors, such as heme oxy- Drp1 attenuates proximal tubule mitochondrial dysfunction and genase 1 (HO-1).22 Conversely, HO-1 can activate NRF2.23 acute tubular necrosis, allowing for cell survival and proliferation Nrf2 and Ho1 transcript levels were increased in Drp1WT in protection against IRI. kidneys at 6 hours, and although Nrf2 transcript levels decreased at 24 hours (Figure 3C), Ho1 levels remained elevated The Renoprotective PGC1a Pathway Is Activated in PTKO (Figure 3D). This response was attenuated in Drp1PTKO kid- Drp1 Mice neys. NRF2 protein (95–110 kD)24 levels are similarly elevated PGC1a, a master regulator of mitochondrial biogenesis, is in IRI kidneys of Drp1WT and Drp1PTKO relative to respective necessary for renal recovery.12,14 NAD can be produced by controls at 24 hours (Figure 3E). Results indicate that the ox- PGC1a-driven biosynthesis or from NAM via the nicotin- idative stress response to IRI is activated in Drp1WT kidneys amide phosphoribosyltransferase (NAMPT) salvage pathway. and to a lesser extent, in Drp1PTKO kidneys. DRP1 mediates High NAD levels leads to accumulation of the fatty acid mitochondrial fission in response to cellular stress and pro- breakdown product b-hydroxybutyric acid and production motes mitochondrial dysfunction. To assess mitochondrial of the renoprotective prostaglandin PGE2. Inhibition of function, primary tubule epithelial cells were isolated from b-hydroxybutyric acid signaling with mepenzolate bromide unoperated Drp1WTand Drp1PTKO mice, and oxidative metab- (MPN) exacerbated kidney injury in mice overexpressing olism was analyzed. Drp1 knockout epithelial cells had PGC1a.12 PGC1a can induce expression of Sirt3, and SIRT3 increased basal oxygen consumption rate, ATP production can indirectly regulate expression of Pgc1a.25 To determine if maximal respiration, and spare reserve capacity compared this pathway is preserved in Drp1PTKO mice, we first measured with WT cells (Figure 3F). These parameters indicate that transcript levels of Nampt, Pgc1a,andSirt3. Pgc1a and Sirt3 were

from kidneys of uninjured mice and cultured for 7 days: (a) oligomycin, (b) carbonyl cyanide-4-(triﬂuoromethoxy) phenylhydrazone, and (c) rotenone and antimycin A. Data in F are representative of three independent experiments and means of triplicate wells 6SD. (G) Rep- resentative images of Mitotracker staining and anti-Tom20 detection and quantiﬁcation of mitochondrial fragmentation in primary tubule epithelial cells. Scale bar, 10 mM. Con, control (no surgery); s, sham. *P,0.05; **P,0.01; ***P,0.001; ****P,0.001.

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Inhibition of DRP1 or Proximal Tubule Deletion of Drp1 after IRI Reduces Kidney Injury and Fibrosis The importance of mitochondrial dynamics in recovery from kidney injury and prevention of fibrosis is not understood. To induce conditional deletion of Drp1 in proximal tubule cells in a time-dependent manner, we generated an inducible, proximal tubule (S1, S2 segment)–specific Drp1 knockout mouse by crossing an iSLC34a1- CreERT2 mouse (tamoxifen-inducible Cre under the control of the SLC34a1 pro- fl fl moter)27 with the Drp1 / mouse to pro- fl fl duce littermate iSLC34a1CreERT2 Drp1 / (iDrp1PTKO)andiSLC34a1CreERT2 Drp1wt/wt Figure 4. Proximal tubule Drp1 knockout results in epithelial cell recovery after IRI. (iDrp1WT) control mice. To assess the effi- Drp1WT and littermate Drp1PTKO mice were subjected to bilateral IRI or sham oper- ciency and location of active Cre-mediated ation and euthanized after 6 or 24 hours of reperfusion. (A) Representative images of loxp site recombination in the kidney, the fl fi immuno uorescence microscopy at 24 hours. (B) Quanti cation of TUNEL+ epithelial iSLC34a1CreERT2 mouse was crossed to a cells in IRI kidneys at 6 and 24 hours or Ki67+ epithelial cells in sham and IRI kidneys at Rosa26-floxed stop tdTomato Cre-reporter 24 hours. Auto, green autofluorescence of kidney tissue with intensity adjusted to fi , mouse. After tamoxifen administration, na- reveal tubule architecture; hpf, high-powered eld. Scale bars, 50 mm. **P 0.01; fl ***P,0.001. tive tdTomato uorescence was observed in proximal tubule CD13+ cells in the cortex (Figure 6A), consistent with previous re- downregulated in kidneys of control mice after IRI (Figure 5A) ports.27 Next, we tested the hypothesis that DRP1 in cortical prox- as previously reported26; however, transcript levels in IRI kid- imal tubule cells is necessary for tissue recovery in the IRI model of neys of Drp1PTKO mice were similar to sham levels (Figure 5A). CKD. iDrp1WT and iDrp1PTKO mice were subjected to 26 minutes Transcript levels of Nampt were increased in IRI kidneys of of unilateral kidney ischemia, and Kim1 transcript levels were Drp1PTKO mice compared with Drp1WT. To test activation of measured at 24 hours of reperfusion, before tamoxifen adminis- this protective pathway in Drp1PTKO mice, MPN or vehicle was tration and induction of Drp1 deletion, to ensure that iDrp1WT administered to Drp1WT and Drp1PTKO mice 1 hour before IRI. and littermate iDrp1PTKO mice had equivalent initial injury. Kim1 As shown in Figure 5B, MPN reversed the protective effect levels were similarly increased in IRI kidneys from iDrp1WT and observed in Drp1PTKO and not observed in Drp1WT mice sub- iDrp1PTKO mice (84.04622.21, n=5 and 57.61622.21, n=4, respec- jected to IRI, suggesting that loss of Drp1 enables proximal tively) compared with the contralateral control kidneys (1.3860.24 tubule cells to activate this protective signaling pathway. and 1.2660.51, respectively). In a separate group of mice,

Figure 5. The renoprotective PGC1a pathway is activated in proximal tubule–speciﬁc Drp1 knockout mice. (A) Drp1WT and littermate Drp1PTKO mice were subjected to bilateral IRI or sham operation and euthanized after 24 hours of reperfusion. Kidney Nampt, Sirt3, and Pgc1a relative to Gapdh transcript levels. (B) PCr in Drp1WT and littermate Drp1PTKO mice that received vehicle (Veh; saline) or MPN (10 mg/kg intraperitoneally), an inhibitor of b-hydroxybutyric acid signaling, 1 hour before bilateral IRI and were euthanized after 24 hours of reperfusion. *P,0.05; **P,0.01; ***P,0.001.

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Figure 6. Delayed deletion of proximal tubule Drp1 or inhibition of DRP1 promotes recovery from IRI. (A) iSLC34a1CreERT2 and iSLC34a1CreERT2 3 tdTomato mice were treated with tamoxifen once daily for 5 days (days 1–5) to selectively induce tdTomato expression in S1/S2 segments of proximal tubule and tdTomato native fluorescence in CD13+ tubules on day 7. (B) Tamoxifen (40 mg/kg intraperitoneally) was administered to iSLC34a1CreERT2 Drp1wt/wt (iDrp1WT)andiSLC34a1CreERT2 Drp1fl/fl (iDrp1PTKO)micebeginning on day 3 after unilateral IRI or sham operation to selectively delete Drp1 in proximal tubule. Mice were nephrectomized (unoperated kidney) on day 13 and euthanized on day 14 for determination of PCr and other end points (for C–G). (C) Contralateral control kidney Drp1 relative to Gapdh transcript levels. (D) PCr and (E) stereologic quantification of renal injury in hematoxylin and eosin–stained sections (F) expressed as a percentage of the total surface area of medullary and cortical portions of the kidney section on day 14. Arrowheads indicate examples of tubular obstruction, atrophy, and dilation. Kidney (G) neutrophil gelatinase–associated lipocalin (Ngal), (H) aquaporin 1 (Aqp1) and solute carrier family 12, member 1 (Slc12a1), and (I) Nrf2 and Ho1 relative to Gapdh transcript levels. (J) Vehicle (saline) or mdivi-1 (50 mg/kg intraperitoneally), an inhibitor of DRP1, was administered to WT C57Bl/6 mice beginning on day 3 after unilateral IRI or sham operation; mice were nephrectomized on day 13, and PCr was determined on day 14. IRI indicates kidney exposed to unilateral ischemia-reperfusion (operated kidney). Auto, green autofluorescence of kidney tissue with intensity adjusted to reveal tubule architecture; con, contralateral control (unoperated kidney); Nx, nephrectomy. Scale bars, 50 mm. *P,0.05; **P,0.01; ***P,0.001; ****P,0.001. tamoxifen was administered at the beginning of the recovery were made 14 days later. Drp1 transcript levels were reduced in phase at the peak of tubule proliferation28 when acute inflamma- iDrp1PTKO compared with iDrp1WT contralateral control whole- tion wanes21 at day 3 of reperfusion (Figure 6B), and assessments kidney tissue lysate (Figure 6C). PCr levels, tubular injury

200 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 194–206, 2018 www.jasn.org BASIC RESEARCH including dilation and atrophy, and expression of neutrophil Table 2. Epithelial tubular structure – PTKO gelatinase associated lipocalin were reduced in iDrp1 mice Sham IRI – WT Tubule compared with controls (Figure 6, D G, Table 2). In some iDrp1 iDrp1WT iDrp1PTKO iDrp1WT iDrp1PTKO mice, proximal tubule cell death was extensive, therefore making 6 6 6 a 6 interpretation of Kim1 results difficult, and thus, they were not Atrophy, % 0.38 0.25 1.39 0.39 40.24 9.36 7.65 3.96 Dilation, % 0.3560.25 0.4060.16 11.5762.94b 4.8861.51 reported. IRI-induced downregulation of proximal tubule (Aqua- c Obstruction, % 0.1760.11 0.2560.11 5.0162.65 1.8360.51 porin 1) and thick ascending limb (solute carrier family 12, member a , WT PTKO + + 2 P 0.001 compared with sham iDrp1 and IRI iDrp1 . 1; coding for the Na -K -2Cl cotransporter) functional genes in bP,0.05 compared with sham iDrp1WT and IRI iDrp1PTKO. control mice was abrogated in iDrp1PTKO mice (Figure 6H). Oxi- cP,0.05 compared with sham iDrp1WT. dative stress, as determined by Nrf2 and Ho1 transcript levels, was attenuated in IRI kidneys of iDrp1PTKO mice compared with con- thermore, DRP1 prevents kidney recovery and promotes kid- trols (Figure 6I). To determine the therapeutic potential of inhibit- ney fibrosis. ing DRP1 after IRI, mdivi-1, an inhibitor of DRP1, or vehicle was Mitochondria are dynamic organelles, and their function administered to wild-type (C57BL/6N) mice every other day be- and morphology are highly coordinated. Prior groups have ginning on day 3 after 26 minutes of unilateral IRI. PCr levels were observed that, within 15 minutes of IRI, mitochondria are attenuated in mice that received mdivi-1 (Figure 6J). Although fragmented in proximal tubules.7,8 We also selected this early mdivi-1 has been widely used as a DRP1 inhibitor, its specificity time point to look at direct effects of Drp1 deletion on mito- has recently come into question.29 chondrial morphology immediately after ischemia rather than We next sought to determine if delayed deletion of Drp1 mitochondrial changes at later time points perhaps due to affected progression to fibrosis after kidney injury. IRI kidneys complex downstream responses to ischemia. Our data show from iDrp1PTKO mice showed decreased fibrotic area as as- that DRP1 mediates this fission event. In mice without sur- sessed by Masson trichrome staining (Figure 6A) and de- gery, mitochondrial morphology does not appear to be differ- creased deposition of large interstitial collagen fibers in the ent with the loss of DRP1, indicating that there must be a cortex and medulla as assessed by picrosirius red morphom- cellular stress to induce DRP1-mediated mitochondrial mor- etry (Figure 6B) compared with iDrp1WT mice. Transcript lev- phologic changes. There was no decrease in mitochondrial els of fibrosis markers (actin, a2; collagen, type I, a1; collagen, area in control mice after IRI, likely due to a combination of type III, a1; fibronectin 1; and vimentin), were decreased in fission and swelling, which was observed in the representative IRI kidneys from iDrp1PTKO mice compared with iDrp1WT EM images. Consistent with the hypothesis that DRP1 medi- mice (Figure 6C). Collagen, type I, a1 and collagen, type III, ates fission after IRI, the decrease in mitochondrial length a1 levels were increased in IRI relative to contralateral control (feret maximum) after IRI in Drp1WT mice is attenuated in kidneys in iDrp1PTKO mice, suggesting that there may be acti- Drp1PTKO mice. Interestingly, there was an increase in area, vation of a wound-healing response in mice that recovered perimeter, and width (feret minimum) but no change in over- from IRI. Cellular mechanisms of fibrosis include myofibro- all shape after IRI in Drp1PTKO mitochondria, suggesting that, blast formation, macrophage accumulation, and peritubular without fission, there may be a shift toward fusion. Likely, microvascular loss.3,30,31 There was a marked decrease in the mitochondrial biogenesis is not occurring at this early time density of PDGFRb+ aSMA+ myofibroblasts in the cortex point, and we are unable to assess clearance. and medulla of IRI kidneys from iDrp1PTKO mice compared Mitochondrial dynamics and total mass are the sum of with controls (Figure 6D, Supplemental Figure 3 shows full fission, fusion, biogenesis, and mitophagy. In this study, mi- sized images). There was also decreased density of F4/80+ in- tochondrial mass was increased in kidneys of control mice at 6 terstitial macrophages, and peritubular capillaries were pre- hours, and it was similar to baseline levels at 24 hours of re- served in IRI kidneys from iDrp1PTKO mice compared with perfusion, which may be due to increased biogenesis or re- iDrp1WT mice (Figure 6D). Additionally, there was attenuated duced mitophagy. In kidneys of Drp1PTKO mice, mitochondrial inflammation, including neutrophils and CD4+ T cells, in IRI mass was unchanged. Because IRI kidneys from Drp1PTKO kidneys from iDrp1PTKO mice compared with iDrp1WT mice mice also have reduced oxidative stress and mitochondrial (Figure 7D). Altogether, our data suggest that proximal tubule swelling, data suggest that, potentially, mitochondrial homeo- DRP1 mediates mitochondrial dysfunction and potentiates mal- stasis is maintained. adaptive repair processes after kidney injury leading to fibrosis. DRP1 can contribute to programmed cell death pathways, such as caspase-dependent apoptosis, as well as caspase- independent regulated necrosis.32,33 Here, we observe similar DISCUSSION TUNEL+ staining, which is typically a marker of apoptosis but can also be observed in other programmed cell death path- This study provides evidence that DRP1 mediates mitochon- ways,33 at 6 hours of reperfusion in Drp1WT and Drp1PTKO drial fission and that, in response to IRI, it mediates dysfunc- kidneys. In contrast, ATN is reduced at 6 hours with the loss tion in proximal tubule cells and promotes IRI-induced cell of DRP1; thus, loss of Drp1 may be limiting regulated necrosis. death and inflammation and reduced kidney function. Fur- Indeed, regulated necrosis has recently emerged as an

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Figure 7. Delayed deletion of proximal tubule Drp1 prevents fibrosis after IRI. Kidneys from iDrp1WT and iDrp1PTKO are the same as in Figure 6B. (A) Representative images of kidney cortex and stereologic quantification of Masson trichrome staining in contralateral control (Con) or unilateral IRI-operated (IRI) kidneys expressed as a percentage of the total surface area of the kidney section. Scale bar, 50 mm. (B) Representative images of picrosirius red staining of kidneys using polarized microscopy and quantification of red/yellow birefringence of mature collagen fibers expressed as a percentage of the total surface area of the cortical and medullary portions of the kidney section. Scale bar, 50 mm. (C) Transcript levels of fibrosis markers by quantitative RT-PCR (relative to Gapdh)atday14.(D) Detection of myofibroblasts (pink) by colocalization of aSMA (blue), PDGFRb pericytes/fibroblasts (red), CD31+ endothelial cells, F4/80+ macrophages, 7/4+ neutrophils, and CD4+ T cells by immunofluorescence microscopy. Acta2/aSMA, actin, a2, smooth muscle, aorta; Auto, green autofluorescence of kidney tissue with intensity adjusted to reveal tubule architecture; Col1a1, collagen, type I, a1; Col3a1, collagen, type III, a1; Fn, fibronectin; Vim, vimentin. Scale bar, 25 mm. *P,0.05; **P,0.01; ***P,0.001; ****P,0.001. important pathway in AKI.34 The mechanisms of regulated triggering necrotic cell death.36–38 In this study, there was no necrosis and specific pathways are unclear and likely, cell and increase in Drp1 transcript levels after IRI, and likely, DRP1 is context dependent. During necroptosis, receptor (TNFRSF)- activated by dephosphorylation or other post-translational interacting serine-threonine kinase 1 and 3 is activated and modifications, as previously shown.16 In other studies, DRP1 leads to phosphorylation of mixed lineage kinase domain like was not involved in necroptosis.39 Mitochondrial permeability and phosphoglycerate mutase family member 5 recruitment to transition (MPT) pore can be opened by Ca2+ overload and mitochondria. Phosphoglycerate mutase family member 5 CypD, allowing for a sudden exchange of solutes and contrib- then recruits and activates DRP1, leading to mitochondrial uting to regulated necrosis in IRI.33,34 The precise molecular fragmentation and cell death.35 Various stimuli, including is- signaling pathways of MPT-mediated regulated necrosis are un- chemia, can activate DRP1-mediated mitochondrial fragmentation, clear in renal proximal tubule cells.

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Regulated necrosis, unlike apoptosis, releases DAMPs and is DRP1 in a diabetic nephropathy model is protective against proinflammatory, a process termed necroinflammation. Fur- kidney disease progression.42 Here, we show that genetic de- thermore, inflammation can trigger regulated cell death in an letion of Drp1 in proximal tubule cells after IRI abrogated autoamplification loop.34 In our study, renal pathology 6 kidney dysfunction and injury. Moreover, the genetic model hours after IRI includes moderate ATN, some innate immune of deletion also showed reduced fibrotic area and hallmarks of cell infiltration of neutrophils and monocytes, and 100- to fibrosis, including myofibroblast formation, extracellular ma- 1000-fold expression of Ccl2 and Il6. At 24 hours, ATN and trix deposition, inflammation, capillary loss, and oxidative innate inflammation are substantial, and chemokine and cy- stress. Given these observations, we anticipate that the poten- tokine expression has declined. Drp1 deletion resulted in re- tial mechanism with the delayed deletion of Drp1 after IRI is duced ATN and neutrophil numbers, pointing to its role in similar to acute studies in that, as AKI progresses to CKD, reducing necroinflammation. In this study, Ccl2 was similarly there is increased proximal tubule mitochondrial dysfunction, elevated at 6 and 24 hours in control and proximal tubule Drp1 fission, oxidative stress, and programmed cell death extending knockout. Consistent with the neutrophil data, Il6 was re- from the medulla to the cortex. In the case of delayed deletion duced at 24 hours with Drp1 deletion. Altogether, these data of Drp1, mitochondrial fission is inhibited, reducing oxidative suggest that DRP1 and mitochondrial fission do not regulate stress, tubular atrophy, and fibrosis. Interestingly, the chemokine expression or that a cell type other than proximal SLCCreERT2 construct does not target the medulla, the area tubules expresses CCL2 and IL6. susceptible to IRI. Thus, we attribute protection to the loss of Determining pathways for proximal tubule cell survival after Drp1 in the S1/S2 segments of the cortex. These studies de- AKI is key for targeting therapies for renal recovery and preven- cisively define a role for DRP1 in ischemic kidney injury and tion of fibrosis. These results provide evidence that, by deleting recovery. Prior studies using mdivi-1 to study the role of DRP1 Drp1 in the proximal tubule, mitochondrial structure and func- may need to be reconsidered given a recent study showing that tion are preserved, renoprotective NAD biosynthesis is activated, it inhibits complex I of the electron transport chain.29 Our and the tubule epithelium exhibits increased proliferation and data suggest that DRP1 may be an important target to promote reduced programmed cell death. Additionally, Pgc1a, a critical recovery after IRI and prevent fibrosis. The translational po- inducer of mitochondrial biogenesis, and Sirt3 expression levels tential for these findings implicates inhibition of DRP1 after are preserved. SIRT3 is an NAD-dependent deacetylase neces- an AKI event as an attractive therapeutic target. sary for maintaining mitochondrial function.25 In the absence of DRP1 and fission, mitochondrial dynamics may tip toward fusion in response to ischemia/ATP depletion and dilute damaged CONCISE METHODS mitochondria, preventing MPT and reducing programmed cell death. In this context, AMPK, a sensor of energy deprivation, Mice may be activated and induce SIRT3 and PGC1a, which induce Experiments were performed in accordance with the National Institutes ROS detoxifying pathways.26,40 Thus, through these cytoprotec- of Health Guide for Care and Use of Laboratory Animals, and all pro- tive pathways, cells may be diverted from a programmed cell cedureswereapprovedbytheUniversityofVirginiaAnimalCareandUse death pathway to survival. Although the specific molecular Committee. Eight- to 12-week-old 18–20 g male mice on a pure C57BL/ events in kidney epithelial cells that link this pathway to pro- 6 background (at least ten generations of backcrossing) were used unless liferation and regeneration are currently unknown, it may be otherwise indicated. PepCKCre mice (a gift from V. Haase, Vanderbilt that, with less programmed cell death, there are more cells to University43; previously described44) were bred to the previously de- respond to the injury stimulus to proliferate. Thus, in the ab- scribed18 (R.J.W. and Z.Y., unpublished data) CAG-CAT-MitoTimer sence of DRP1, the cell likely has the metabolic capacity to shift transgenic mice (a gift from Z.Y., University of Virginia) to produce from a programmed cell death pathway to a survival and pro- PepCKCre+ MitoTimer+ mice. PepCKCre mice were also bred to mixed liferation pathway. The observation of increased proliferation in background Drp1f/f mice (H.S., Johns Hopkins University)45 to generate Drp1PTKO kidneys is an apparent discrepancy with previous find- PepCKCre+Drp1f/f and littermate PecpCKCre2 Drp1f/f mice. iSLC34a1- ings that the proximal tubule–specificdeletionofMfn2, a me- CreERT2 mice (provided by B. Humphreys, Washington University)27 diator of fusion, also increases tubule proliferation 24 hours after were bred to (Gt)Rosa26Sor floxed-stop tdTomato reporter mice IRI.41 However, proximal tubule–specific deletion of Mfn2 did (Ai9 mice; Jackson Laboratory) or Drp1f/f mice to produce iSLC34a1- not result in altered apoptosis, necrosis, or inflammation. This CreERT2 3 tdTomato, iSLC34a1CreERT2 Drp1f/f, and littermate iSL- suggests that epithelial programmed cell death versus prolifera- C34a1CreERT2 Drp1wt/wt. C57BL/6 mice were purchased from the tion in response to ischemia is more than a simple tipping the NCI Mouse Repository. Mice were maintained in standard vivarium balance of fission and fusion, and it may also depend on protein- housing with a 12-hour light/dark cycle on a chow diet, and water was protein interactions and cell signaling events. freely available. Tamoxifen (T5648; Sigma-Aldrich) was dissolved in 5% Antioxidant agents and mitochondrial-targeted molecules EtOH in USP corn oil and administered intraperitoneally to all mice to reduce mitochondrial dysfunction and oxidative stress have at 40 mg/kg per day as described elsewhere.46 MPN (M5651; Sigma- been proposed as a therapeutic strategy in CKD.6 Prevention of Aldrich) was dissolved in saline and administered intraperitoneally at podocyte mitochondrial dysfunction through inhibition of 10 mg/kg 1 hour before surgery.

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Surgery: IRI Electron Microscopy Mice were anesthetized with an intraperitoneal injection of a keta- Mice were subjected to 26 minutes of bilateral ischemia, and after 15 mine (120 mg/kg) and xylazine (12 mg/kg) mixture and placed on a minutes of reperfusion, they were reanesthetized for perfusion fixation. warm pad to maintain body temperature at 34.5°C–36°C. Mice were Briefly, mice were perfused transcardially with 5 ml heparinized (10 U/ml) then randomized to sham or IRI operation. Bilateral or unilateral saline followed by 20–25 ml freshly prepared 4% paraformaldehyde/2.5% flank incision was performed, and the renal vessels (vein and artery) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Kidneys were on either both sides or only the left side were crossclamped for 26 removed, and 1- to 2-mm horizontal slices were postfixed overnight in the minutes. Sham-operated mice underwent the same procedure, with same fixative; one section was postfixed in 4% paraformaldehyde without the exception of vessel clamping. Surgical wounds were closed, and glutaraldehyde and subsequently processed for hematoxylin and eosin buprenorphine (0.15 mg/kg intraperitoneally) was administered as staining. The remaining sections were then rinsed three times in 0.1 M an analgesic. In unilateral IRI experiments, mice were reanesthetized phosphate buffer (pH 7.4), and they were prepared for electron micros- and subjected to right kidney nephrectomy 24 hours before eutha- copy by incubating in 2% osmium (in 0.1 M phosphate buffer [pH 7.4]) nasia to reveal changes in kidney function of the operated ischemic for 1 hour, washing and dehydrating through a graded series of ethanol, (left) kidney assessed by measuring PCr. Mice were euthanized by and embedding in EmBed812 embedding resin (Electron Microscopy overdose with the ketamine/xylazine mixture and cervical disloca- Sciences, Fort Washington, PA). Serial ultrathin sections (80–90 nm) of tion for confirmation of death. Both the ischemic (IRI) and unop- kidney cortex were collected on copper mesh grids and stained with erated (nephrectomized; contralateral control) kidneys were saved for uranyl acetate and Reynolds lead citrate. Two to three grids from each analysis. sample were examined using a JEOL 1230 transmission electron microscope, and digital photographs of 8–15 proximal tubules were captured by Assessment of Kidney Function and Histology real-time digital imaging. Blood was collected under anesthesia from the retro-orbital sinus, and PCr (milligrams per deciliter) was determined by using an enzymatic Ultrastructural Analyses fi ’ method with minor modi cations from the manufacturer s protocol Electron micrographs were opened in Stereo Investigator software, which (twice the volume of sample; Diazyme Laboratories) as previously was calibrated to match the scale of the images, and all mitochondrial 47 described. Hematoxylin and eosin staining and stereologic analysis contours within the photographed proximal tubule (yielding 25–100 48 were performed as previously described. Masson trichrome was mitochondrial contours per tubule) were drawn to obtain an unbiased 49 performed and analyzed as previously described. For picrosirus estimate of mitochondrial morphology. Contour measurements (feret fi red staining, kidneys were xed in 4% paraformaldehyde/0.1 M so- minimum and maximum, area, perimeter, aspect ratio, shape factor, dium phosphate buffer (pH 7.4) overnight, changed to 70% ethanol, and roundness) were automatically calculated using embedded equations fi subsequently embedded in paraf n, and sectioned at 4-mm thickness. within the software. Feret maximum (the longest projection of the min- fi Sections were deparaf nized, rehydrated, and stained in picrosirius imal bounding box; i.e., parallel tangents apposing opposite sides of the ’ red solution according to the manufacturer s protocol (Polysciences). profile) and minimum (the shortest projection of the minimal bounding Photomicrographs of picrosirius red birefringence were captured box), area, perimeter, aspect ratio (describes the degree of flatness of a fi using a Zeiss AxioImager microscope with polarizing lter (Carl Zeiss contour; range of values is zero to one; a circle has a value of one), 3 GmbH) at 200 and Stereo Investigator software (version 9; MBF roundness(valuesarezerotoone;acirclehasavalueofone),shapefactor fi fi Bioscience). Quanti cation of the brotic area was done by averaging (a contour with a large shape factor has a convoluted outline; a circle has fi fi total red/yellow birefringent pixels of ve random elds per kidney thesmallestshapefactoratavalueof3.54)arereported: section with ImageJ software (http://imagej.nih.gov/ij/). MaxDiameter Aspect Ratio ¼ Quantitative Real-Time PCR MinDiameter Perimeter Total RNA was isolated and reversed transcribed to cDNA, and RT- Shape factor ¼ pffiffiffiffiffiffiffiffiffi PCR was performed as previously described.49 Primers (Supplemen- Area 4zArea tal Table 1) span an exon-exon junction and were designed with Roundness ¼ : Primer-BLAST (NCBI). For mitochondrial mass, genomic DNA pzMaxDiameter2 was purified from whole-kidney tissue lysates, and the ratio of mito- 9 9 chondrial 16S (For 5 -CCGCAAGGGAAAGATGAAAGAC-3 ,Rev Mitochondrial Respiration 59-TCG TTT GGT TTC GGG GTT TC-39) to nuclear EEF1A1 (For Primary tubule epithelial cells were isolated from the kidney as previously 59-GGATTG CCA CAC GGC TCA CAT T-39,Rev59-GGT GGATAG described.50 Tubule epithelial cells were grown to confluence on Seahorse TCT GAG AAG CTC TC-39) DNA was determined. XF analyzer (Agilent, Santa Clara, CA) 24-well tissue culture plates; oxygen consumption rate was measured, and parameters were calculated as Flow Cytometry previously described50 with the following modification. Mitochondrial Kidney single-cell suspensions were prepared, labeled, and analyzed as respiration inhibitors were used at the following concentrations: oligo- previously described.47 Ly6G (2 mg/ml; clone 1A8), CD11b (2 mg/ml; mycin (2 mM; Sigma-Aldrich), carbonyl cyanide-4-(trifluoromethoxy) clone M1/70), and CD45 (1 mg/ml; clone 30F11) were purchased phenylhydrazone (1.5 mM; Sigma-Aldrich), and rotenone (0.1 mM; from eBioscience. Sigma-Aldrich) with antimycin A (2 mM; Sigma-Aldrich).

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Immunofluorescence Research conducted for this publication was supported by National Kidney samples were prepared and labeled, and images were acquired and Institute of Diabetes and Digestive and Kidney Diseases of the National quantified as previously described.47 Goat anti-CD13 A647 (R&D Sys- Institutes of Health awards F32DK108563 (to H.M.P.), T32DK72922 (to tems), rabbit anti-Ki67 (clone SP6; Vector Laboratories) or rabbit anti- H.M.P.), American Society of Nephrology Ben Lipps Fellowship Program Tom20 (polyclonal; Santa Cruz Biotechnology) and donkey anti-rabbit (to H.M.P.), DK091444 (to A.B.), DK107941 (to A.B.), R01 062324 (to Cy3 (Jackson ImmunoResearch Laboratories), rat anti-CD31 A647 (clone M.D.O.), and R01 085259 (to M.D.O.). The stereology data described here MEC13.3; BioLegend), rat anti-F4/80 A555 (clone BM8; Invitrogen), rat were gathered on an MBF Bioscience and Zeiss microscope system for antineutrophil FITC or PE (clone 7/4; Cedarlane), rat anti-CD4 A647 stereology and tissue morphology funded by National Institutes of Health (clone GK1.4; Invitrogen), rabbit anti-aSMA Cy3 (clone 1A4; Sigma- grant 1S10RR026799-01 (to M.D.O.). Electron micrographs were ob- Aldrich), and rat anti-PDGFRb A647 (clone APB; BioLegend) were tained by using a JEOL Model JEM-1230 Transmission Electron Micro- used at 2 mg/ml for primary antibodies and 1.5 mg/ml for the secondary scope funded by National Institutes of Health grant 1S10RR021017-01. antibody. TUNEL assay (In Situ Cell Death Detection kit; TMR red; The content is solely the responsibility of the authors and does not Roche) was performed according to the manufacturer’s instructions. necessarily represent the official views of the National Institutes of Data are means6SEM of five high-powered fields per kidney section Health. per mouse. For PepCKCre MitoTimer mice, ratiometric analysis was performed as previously described.18 Data are means6SEM of five to eight fi high-powered elds of the outer medulla per kidney section per mouse. DISCLOSURES Primary epithelial cells were stained with 100 nM Mitotracker red M.D.O. served on an advisory board for Mitobridge, Inc. CMXRos (Invitrogen) for 45 minutes. Cells were then washed and al- lowed to recover in media; then, they were fixedandmountedwithPro- Long Gold Antifade with DAPI (Invitrogen). 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