Sphingosine 1-Phosphate Receptor-1 Enhances Mitochondrial Function and Reduces Cisplatin-Induced Tubule Injury

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† †‡ † † Amandeep Bajwa,* Diane L. Rosin, Piotr Chroscicki,* Sangju Lee,* Krishna † † † † † Dondeti,* Hong Ye,* Gilbert R. Kinsey,* Brian K. Stevens,* Katarzyna Jobin,* ‡ ‡ ‡ † Brandon M. Kenwood, Kyle L. Hoehn, Kevin R. Lynch, and Mark D. Okusa*

Departments of *Medicine and ‡Pharmacology, and †Center for Immunity, Inﬂammation, and Regenerative Medicine, University of Virginia School of Medicine, Charlottesville, Virginia

ABSTRACT Sphingosine 1-phosphate (S1P), the natural sphingolipid ligand for a family of five G protein– coupled receptors (S1P1–S1P5Rs), regulates cell survival and lymphocyte circulation. We have shown that the pan- S1PR agonist, FTY720, attenuates kidney ischemia-reperfusion injury by directly activating S1P1 on proximal tubule (PT) cells, independent of the canonical lymphopenic effects of S1P1 activation on B and T cells. FTY720 also reduces cisplatin-induced AKI. Therefore, in this study, we used conditional PT-S1P1-null fl/fl w/wt (PepckCreS1pr1 ) and control (PepckCreS1pr1 ) mice to determine whether the protective effect of FTY720 in AKI is mediated by PT-S1P1. Cisplatin induced more renal injury in PT-S1P1-null mice than in controls. Although FTY720 produced lymphopenia in both control and PT-S1P1-null mice, it reduced injury only in control mice. Furthermore, the increase in proinflammatory cytokine (CXCL1, MCP-1, TNF-a, and IL-6) expression and infiltration of neutrophils and macrophages induced by cisplatin treatment was attenuated by FTY720 in control mice but not in PT-S1P1-null mice. Similarly, S1P1 deletion rendered cultured PT cells more susceptible to cisplatin-induced injury, whereas S1P1 overexpression protected PT cells from injury and preserved mitochondrial function. We conclude that S1P1 may have an important role in stabilizing mitochondrial function and that FTY720 administration represents a novel strategy in the prevention of cisplatin-induced AKI.

J Am Soc Nephrol 26: 908–925, 2015. doi: 10.1681/ASN.2013121351

Cisplatin is a platinum-based chemotherapeutic onrenalepithelialcells11 and in vivo studies of drug used to treat various types of cancers.1 Neph- kidney ischemia-reperfusion injury (IRI)12,13 rotoxicity, which occurs in about one-third of demonstrated a role of sphingolipids in AKI. The patients undergoing cisplatin treatment,2,3 often limits the use of cisplatin4; management of cisplatin- Received December 28, 2013. Accepted June 29, 2014. induced AKI rests primarily with decreasing the dose of cisplatin, thereby putting chemotherapeutic efﬁcacy Published online ahead of print. Publication date available at www.jasn.org. at risk. Renal proximal tubule (PT) cells, a primary site Present address: Dr. Sang Ju Lee, Division of Nephrology, De- partment of Internal Medicine, Daejeon St. Mary’sHospital, of cisplatin toxicity, actively accumulate cisplatin Catholic University of Korea, Daeheungdong, Chungku, Daejeon 5–8 through three transporters. Like other forms of 301-723, South Korea. AKI, cisplatin nephrotoxicity encompasses direct Correspondence: Dr. Amandeep Bajwa, Division of Nephrology, 8 ﬂ cellular as well as in ammatory and immune Department of Medicine, University of Virginia Health System, mechanisms, including elevated cytokines/chemo- Box 800746, Charlottesville, VA 22908. Email: ab9nh@virginia. kines and immune cell recruitment,9,10 that result edu from and may potentiate cell death. In vitro studies Copyright © 2015 by the American Society of Nephrology

908 ISSN : 1046-6673/2604-908 J Am Soc Nephrol 26: 908–925, 2015 www.jasn.org BASIC RESEARCH similarity in pathophysiology between cisplatin-induced injury and other models of AKI led us to consider a potential protective role of sphingosine 1-phosphate (S1P) in cisplatin- induced AKI. S1P, the ligand for five G protein–coupled receptors (S1P1R– S1P5R), evokes diverse cellular signaling responses.14–16 FTY720, through its active phosphorylated form, is a non- selective S1P agonist at S1P1 and SIP3–517,18 used in the treatment of multiple sclerosis.19 S1PR activation promotes cell survival.20,21 In various disease models, FTY720 causes reversible redistribution of lymphocytes from the circulation to secondary lymph tissue leading to its anti-inflammatory and tissue-protective effects.17,22 FTY720 protects kidneys from IRI through PT-S1P1 activation.12,13 S1P1 activation by various means, including S1P agonists SEW2871 and FTY720, decreases apoptosis and enhances cell survival in response to a variety of stressors23,24 by preventing mitochondrial dysfunction through mechanisms including decreased cytochrome c release,25 BAX translocation,26 and regulation of bcl-2 proteins.27 We hypothesized that FTY720 would reduce cisplatin- induced nephrotoxicity by directly targeting PT-S1P1. We demonstrate that S1P1 plays a critical role in improving mitochondrial function and rendering PT epithelial cells resistant to the apoptotic/necrotic effects of cisplatin. Figure 1. WT mice treated with FTY720 are protected from cisplatin-induced AKI. WT mice were treated with FTY720 (240 mg/kg) RESULTS 1hourbeforeasingledoseofcisplatin(27mg/kg)andonceeachday on the next 2 days. (A) Plasma creatinine 72 hours after cisplatin. FTY720 Attenuates Inflammation and Cisplatin- Vehicle, n=4; cisplatin, n=8; cisplatin+FTY720, n=11. (B) H&E staining Induced AKI of kidney sections from the same mice. Insets show a 32.5 magnified Treatment of wild-type (WT) mice with cisplatin caused a image. (C) Semiquantitative analysis of tubular injury from H&E- – , , significant rise in plasma creatinine at 72 hours compared stained kidney sections scored on a scale of 0 5. *P 0.05; **P 0.01; , 6 with vehicle-treated mice, and FTY720 attenuated this re- ***P 0.001. Values are the mean SEM. H&E, hematoxylin and eosin. Bar, 100 mm. duction in renal function (Figure 1A). Kidneys of cisplatin- treated mice had increased acute tubular necrosis (ATN) compared with vehicle-treated mice, and FTY720 reduced cisplatin treatment alone (Figure 2). Proinflammatory cytokine cisplatin-induced injury (Figure 1B). Histologic assessment and chemokine mRNA levels increased significantly in kidney of tubule injury (ATN score) paralleled functional data after cisplatin treatment compared with vehicle-treated (Figure 1C). Compared with vehicle-treated mice, cisplatin treatment resulted in increased macrophages and neutrophils primarily fl fi Table 1. Proin ammatory cytokine and chemokine in the outer medulla, and FTY720 reduced this cell in ltration expression in kidneys of WT mice treated with cisplatin or compared with cisplatin alone (Supplemental Figure 1), as cisplatin+FTY720 demonstrated by flow cytometry. Immunofluorescence la- Cisplatin Cisplatin+FTY720 beling of kidney sections revealed more neutrophil (7/4) 6 a 6 and monocyte (F4/80) infiltration and more CXCL1 and CXCL1 60.9 26.4 13.7 6.9 MCP-1 83.1630.9a 18.266.7 MCP-1 immunoreactivity (note MCP-1 immunofluores- IL1-b 12.364.3a 1.260.7b cence in PT consistent with increased mRNA levels; Table 1) IL-6 216.9674.2a 62.7624.1b after cisplatin treatment; FTY720 reduced these effects TNF-a 77.7627.4a 28.7612.3 (Figure 2). As expected, some neutrophils in injured kidney Doses of cisplatin and cisplatin+FTY720 as in Figure 1. mRNA expression (72 were immunoreactive for CXCL1.28 Administration of FTY720 hours after cisplatin) relative to glyceraldehyde 3-phosphate dehydrogenase also preserved the endothelium (CD31) and reduced apoptosis expressed as fold changes compared with vehicle. Values are the – mean6SEM. n=6–8 in each group. (terminal deoxynucleotidyl transferase mediated digoxigenin- aP,0.05 compared with vehicle. deoxyuridine nick-end labeling [TUNEL]) compared with bP,0.05 compared with cisplatin.

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Concise Methods). Cisplatin produced significantly more renal injury as measured by fl/fl plasma creatinine in PepckCreS1pr1 than in wt/wt PepckCreS1pr1 (control). FTY720 reduced cisplatin-induced nephrotoxicity in wt/wt fl/fl PepckCreS1pr1 but not in PepckCreS1pr1 (Figure 3A). Morphologic changes (Figure 3B) paralleled functional studies (ATN wt/wt scores: PepckCreS1pr1 cisplatin 2.8960.22 versus cisplatin+FTY720 1.6660.21; fl/fl PepckCreS1pr1 cisplatin 3.0860.21 versus cisplatin+FTY720 3.5660.28). As expected,12,13 FTY720 produced lymphopenia fl/fl wt/wt in PepckCreS1pr1 and PepckCreS1pr1 mice (data not shown). These data demonstrate that PT-S1P1 deficiency enhanced cisplatin-induced injury and that the protective effect of FTY720 in cisplatin-treated mice, as in mice exposed to IRI,12 is independent of lymphopenia but dependent on expression of S1P1 in PT cells. FTY720 reduced the cisplatin-induced increase in the number of macrophages wt/wt and neutrophils in PepckCreS1pr1 kid- fl/fl neys but was ineffective in PepckCreS1pr1 mice (Supplemental Figure 2). Similarly, the cisplatin-induced increase in neutrophils, monocytes, CXCL1 expression (Supplemen- tal Figure 2), and apoptosis (TUNEL) was wt/wt reduced by FTY720 in PepckCreS1pr1 fl/fl but not PepckCreS1pr1 mice (Figure 3C). Inflammatory markers (mRNA levels) in- wt/wt creased significantly in PepckCreS1pr1 fl/fl and PepckCreS1pr1 kidneys after cisplatin, but FTY720 attenuated the increased wt/wt expression only in PepckCreS1pr1 and not fl/fl in PepckCreS1pr1 mice (Table 2). Incuba- tion with cisplatin ex vivo increased cleaved- Figure 2. FTY720 treatment attenuates leukocyte infiltration and protects kidneys after caspase 3 in a concentration-dependent cisplatin. Same treatments as in Figure 1. Immunofluorescence of kidney sections with manner in primary TEC cultures isolated endothelial cell (CD31; white), neutrophil (7/4; green, or yellow if merged with CXCL1 or PepckCreS1pr1wt/wt MCP-1 labeling [red]), monocyte (F4/80; white), cytokine (MCP-1; red), and chemokine from kidneys, and at lower cisplatin concentrations the cells from (CXCL1; red) labeling and apoptotic cell staining (TUNEL; red). Blue, nuclei stained with fl/fl DAPI. TUNEL staining (red) in DAPI-labeled (blue) nuclei appears magenta in color- PepckCreS1pr1 , which lack S1P1, were merged image. Bar, 50 mm. more susceptible to injury (Figure 3, D–F). mice, and this increased expression was reduced by FTY720 FTY720-Mediated Protection of Cisplatin-Induced PT (Table 1). Cell Injury In Vitro Is Attenuated by S1P1 Deficiency The cisplatin-induced increase in chemokine and proinflamma- PT-S1P1 Deficiency Leads to Enhanced Cisplatin-Induced tory cytokine mRNA expression in TKPTS cells was reduced by Nephrotoxicity and Renders FTY720 Ineffective phosphorylated FTY720 (FTY720-p; Table 3, left). Cisplatin dis- Our in vitro and in vivo deletion studies demonstrate that PT-S1P1 rupted the cytoskeletal structure and increased CXCL1 and MCP- mediates protection from ischemia-reperfusion.13 To investigate 1 immunoreactivity compared with TKPTS treated with vehicle the direct involvement of PT-S1P1 in cisplatin-induced neph- or FTY720-p alone; pretreatment with FTY720-p attenuated these rotoxicity, we used conditional deletion of S1pr1 in PT cells13 effects of cisplatin (Supplemental Figure 3). Cisplatin decreased (see also tubular epithelial cell [TEC] characterization in cell viability (73.1%66.3% of vehicle; P,0.05 relative to vehicle),

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Figure 3. Deletion of PT S1P1R exacerbates injury and its presence is necessary for FTY720-mediated protection from cisplatin-induced AKI. Mice were treated with FTY720 1 hour before cisplatin and once each day on the next 2 days (as described in Figure 1). (A) Plasma creatinine levels of PepckCreS1pr1wt/wt (littermate controls) and PepckCreS1pr1fl/fl mice (deficient in S1P1 on PT epithelial cells) 72 hours after cisplatin. n=5–11. *P,0.05 compared with respective cisplatin; **P,0.01 compared with vehicle; #P,0.05 compared with cisplatin. (B) H&E staining of kidney sections. Insets show a 32.5 magnified image. (C) Immunofluorescence labeling of apoptosis (TUNEL) in kidney sections from PepckCreS1pr1wt/wt and PepckCreS1pr1fl/fl mice. TUNEL labeling is barely detectable in kidney sections from vehicle-treated mice (Supplemental Figure 2). TUNEL staining (red) in DAPI-labeled (blue) nuclei appears magenta in color-merged image. (D and E) Repre- sentative quantitative fluorescence Western blot of caspase 3 (Casp 3) and cleaved-caspase 3 (c-Casp 3) (both Casp 3 and c-Casp 3 revealed with IRdye680RD secondary antibody) and tubulin (revealed with IRdye800CW secondary antibody) in homogenates of primary TEC cultures from PepckCreS1pr1wt/wt and PepckCreS1pr1fl/fl mice after incubations with vehicle (saline) or increasing concentrations of cisplatin for 24 hours. (F) Densitometric analysis of c-Casp 3 fluorescence (normalized to tubulin). Concentrations of cisplatin (indicated on blot and bar graph by widening triangle): 10, 20, 40, or 60 mM. n=2 wells in each of two replicate experiments. Values are the mean6SEM. H&E, hematoxylin and eosin; ND, not detectable; V, vehicle. Bar, 100 mminB;50mminC. whereas FTY720-p, which had no effect alone, preserved cell vi- TKPTS cells transfected with S1p1r-specific small interfering ability (90.963.3% of vehicle; P,0.05 compared with cisplatin RNA (siRNA); S1pr1 mRNA expression was reduced by alone) when cells were pretreated 1 hour before cisplatin. 50%–80% (with no change in S1pr3 expression).13 The cisplatin- Tocomplement our in vivo studies and demonstrate that PT- induced increase in cleaved-caspase 3 in TKPTS cells was S1P1 mediates the protective effect of FTY720, we studied prevented by FTY720-p. S1pr1 deficiency increased basal

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wt/wt fl/fl Table 2. Proinflammatory cytokine and chemokine expression in kidneys of PepckCreS1pr1 and PepckCreS1pr1 mice treated with cisplatin or cisplatin+FTY720 PepckCreS1pr1wt/wt PepckCreS1pr1fl/fl

Cisplatin Cisplatin+FTY720 Cisplatin Cisplatin+FTY720 CXCL1 257.466151.6a 62.466.8 340.16319.2a 378.4683.2 MCP-1 188.26114.1a 22.765.1b 40.7625.8a 87.1616.3 IL-1b 21.7613.1a 3.761.5 18.6611.4a 175.96146.6 IL-6 503.76252.1a 88.3630.6 116.6667.2a 192.2648.8 TNF-a 96.4641.0a 35.569.9 69.1638.5a 51.1616.5 Doses of cisplatin and cisplatin+FTY720 as in Figure 1. mRNA expression (72 hours after cisplatin) relative to glyceraldehyde 3-phosphate dehydrogenase expressed as fold changes compared with vehicle. Values are the mean6SEM. n=3–6ineachgroup. aP,0.05 compared with vehicle. bP,0.05 compared with cisplatin. vehicle-treated and cisplatin-induced levels of cleaved-caspase resistant to concentration-dependent (10–80 mM) cisplatin- 3, but FTY720-p was not protective (Figure 4, A and B); in- induced decreases in cell viability observed in control pcDNA- creasedexpressionwasdependentontheconcentrationof TKPTS (80%–85% of vehicle). Cellular morphology and cisplatin (Supplemental Figure 4, A–C). FTY720-p pretreatment integrity of the cell culture monolayer were disrupted by cis- did not attenuate the cisplatin-induced increase in CXCL1, IL-6, platin in a concentration-dependent manner in control cells and TNF-a expression in TKPTS cells transfected with S1pr1 (Figure 4, Supplemental Figure 3A), but were preserved in siRNA (Table 3, right). S1pr1-TKPTS (Figure 5, A, C, and D, Supplemental Figure In examining the protective effect of S1P1 in cisplatin-induced 5D); cisplatin also increased cleaved-caspase 3 in control cells apoptosis via the intrinsic mitochondrial pathway, we found that in a concentration-dependent manner (Supplemental Figure incubation of TKPTS cells with cisplatin increased expression of 4D). Therefore, 20 mM cisplatin (for 24 hours) was selected for BAX and translocation from the cytoplasm (monomeric form) to additional experiments (Figure 5). mitochondria (polymerized form); as expected,26 these changes To examine mitochondrial function, cellular morphology, were prevented by FTY720-p pretreatment (Figure 4, C–E). and susceptibility to apoptosis, we performed immunofluores- Untreated primary cultures of PT epithelial cells from Pepck- cence labeling of vehicle- and cisplatin-treated pcDNA- and fl/fl CreS1pr1 showed marked disruption of cell structure com- S1pr1-TKPTS (Figure 5). Consistent with BAX translocation to wt/wt pared with cells from PepckCreS1pr1 . Cell injury (disruption mitochondria after cisplatin-induced injury (Figure 4C; and in of normal cell structure and mitochondrial morphology; cell pcDNA-TKPTS, Figure 5E), where it disrupts mitochondrial death) and mitochondrial BAX expression after cisplatin was integrity and allows leakage of cytochrome c, increased cytoplas- fl/fl greater in primary PT cell cultures from PepckCreS1pr1 kid- mic cytochrome c was observed in cisplatin-treated control cells wt/wt neys than from PepckCreS1pr1 (Supplemental Figure 3). (indicative of apoptosis). By contrast, cytochrome c was retained in mitochondria of vehicle-treated pcDNA-TKPTS and vehicle- Overexpression of S1pr1 Stabilizes Mitochondria and or cisplatin-treated S1pr1-TKPTS, where it colocalized with Protects TKPTS Cells from Cisplatin-Induced Injury MitoTracker Red (Figure 5A). Mitochondrial BAX was observed In contrast with S1P1-deficient cells, S1P1 overexpressing in cisplatin-treated pcDNA-TKPTS but not S1pr1-TKPTS (Fig- TKPTS cells were resistant to the effects of cisplatin. S1pr1- ure 5E, Supplemental Figure 5, E and F). Consistent with their TKPTS cells (100%–110% viability relative to vehicle) were resistance to cisplatin-induced cell death, S1pr1-TKPTS cells

Table 3. Proinﬂammatory cytokine and chemokine expression in TKPTS cells (control and after siRNA S1P1 knockdown) treated with vehicle, FTY720-p, cisplatin, or cisplatin+FTY720-p Control siRNA Knockdown Vehicle FTY720-p Cisplatin Cisplatin+FTY720-p Vehicle FTY720-p Cisplatin Cisplatin+FTY720-p CXCL1 1.060.40 1.860.13 21.963.20a 8.261.5b 1.060.01 1.160.08 16.860.88a 17.861.06 MCP-1 1.060.02 0.960.05 3.1960.2 1.960.08 1.060.03 1.26-0.05 2.760.24 2.860.34 IL-1b 1.060.6 14.565.9 186.8698.4a 68.769.9b 1.060.20 1.660.2 6.961.9 12.461.17 IL-6 1.060.5 1.860.64 21.368.8a 16.560.8 1.060.21 1.960.10 126.5613.4a 153.3615.2 TNF-a 1.060.7 1.360.5 13.2610.2a 1.6961.03 1.060.03 1.160.11 21.660.92a 20.760.877 The left columns show data for untreated TKPTS cells, whereas the right columns show data for cells transfected with S1pr1 siRNA 48 hours before the experiment. Control (scrambled siRNA) or knockdown cells were incubated with vehicle (saline) or cisplatin (20 mm) for an additional 24 hours. mRNA expression relative to glyceraldehyde 3-phosphate dehydrogenase expressed as fold changes compared with respective vehicle. Values are the mean6SEM. n=3 in each group. aP,0.05 compared with respective vehicle. bP,0.05 compared with respective cisplatin.

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and F, Supplemental Figure 5, G–J). In addition to the findings with JC-1 and MitoSox that S1P1 overexpression preserved mitochondrial health, labeling with MitoTrackerRed revealed a striking difference in mitochondrial morphology between the two populations of cells. MitoTracker-immunoreactive mitochondria in pcDNA-TKPTS showed normal morphology, but typical of apoptotic cells, ap- peared fragmented after cisplatin treatment. By contrast, MitoTracker immunoreactivity in S1pr1-TKPTS did not appear to change after cisplatin treatment (Figure 5D). Ultrastructural analysis revealed that mitochondria in pcDNA-TKPTS were shorter and rounder (Figure 6A), whereas on average they were longer and thinner in S1pr1-TKPTS (Figure 6B), as confirmed by quantitative assessment of mitochondrial morphology. Analysis of mitochondrial contour measurements (Concise Methods) yielded values for compactness, feret maximum, and shape factor of 0.60960.011, 0.50660.022 and 4.03660.036 for pcDNA-TKPTS (20 cells; 307 mitochondria) and 0.52760.017*, 0.67560.027*, and 4.40060.070* for S1pr1-TKPTS, respectively (25 cells; 346 mitochondria; *P,0.001 versus respective Figure 4. Phosphorylated FTY720 (F-p)-–mediated protection of cisplatin-induced injury value for pcDNA-TKPTS). In contrast with in TKPTS cells is attenuated by S1pr1 siRNA knockdown. (A) Mouse kidney PT (TKPTS) S1pr1 cells were transfected with scrambled or S1pr1 siRNA 48 hours before incubation with stable overexpression, transient knock- vehicle or FTY720-p (10 nM) for 1 hour and then cisplatin (20 mM) for an additional 24 down with siRNA (Figure 6, C and D) or re- hours. Western blot for full-length caspase 3, cleaved-caspase 3 (c-Casp 3), and tubulin peated treatment of TKPTS cells with from TKPTS cells transfected with scrambled or siRNA against S1pr1 (detection as in FTY720 (which can induce receptor internal- Figure 3). Western blot is from a representative experiment with three to four replicates of ization and degradation in immune cells) did each treatment, and the results of three experiments (densitometric analysis) are shown in not alter mitochondrial contour measure- B. (C) Immunofluorescence labeling of actin cytoskeleton (FITC-phalloidin; Actin), apo- ments (Supplemental Table 1). ptotic marker (Bcl-2–associated X protein; BAX White), and mitochondria (MitoTracker m Red; Mito) in TKPTS cells treated with vehicle or cisplatin (20 M for 24 hours) with or S1P1 Overexpression Increases without prior incubation (1 hour) with FTY720-p (10 nM). Blue, nuclei stained with DAPI. Mitochondrial Respiration in PT Cells Overlay of red MitoTracker and white BAX appears pink in merged images. Arrows, cells Analysis of mitochondrial bioenergetics in with increased BAX immunoreactivity. Images are from a representative experiment performed at least three times with two to three replicates of each treatment. (D and E) whole cells, by measuring oxygen consump- Western blot (D) and densitometric analysis (E) of BAX in cytoplasmic and mitochondrial tion over time after the sequential addition of fractionsofTKPTScellstreatedwith vehicle or cisplatin (20 mM for 20 hours) with or without inhibitors of mitochondrial function,29 re- prior incubation (1 hour) with FTY720-p (10 nM). C, cytoplasmic; M, mitochondrial; Cis, vealed marked differences between the two cisplatin. Bar, 10 mm. populations of cells (Figure 7). S1pr1- TKPTS had higher rates of basal mitochon- were less sensitive to the concentration-dependent cisplatin- drial respiration (2.53) than pcDNA-TKPTS, usually indicative induced increases in cleaved-caspase 3 than control cells of either more mitochondria or increased mitochondrial activity (Supplemental Figure 4, D–F). Injured cisplatin-treated (Figure 7, A and B). Vehicle-treated pcDNA- and S1pr1-TKPTS pcDNA-TKPTS but not S1pr1-TKPTS cells showed decreased ac- did not differ in their whole cell respiratory control ratio, the ratio cumulation of the mitochondrial membrane potential–sensitive between the maximal mitochondrial respiration rate induced by; JC-1 (Figure 5B). Concentration-dependent cisplatin-stimulated carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone generation of mitochondrial superoxide, revealed by oxidation- (FCCP) and ATP-linked respiration (Figure 7C), or their cou- dependent increased fluorescence of MitoSox Red indicator, pling efficiency, the ratio of ATP-linked mitochondrial respira- was observed in control but not S1pr1-TKPTS cells (Figure 5, C tion to basal mitochondrial respiration (Figure 7D). Coupling

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Figure 5. Overexpression of S1P1 protects TKPTS cells from cisplatin-induced cell death and mitochondrial injury. TKPTS cells were stably transfected (and maintained under G418 selection) with control pcDNA3.1 vector (pcDNA-TKPTS) or vector containing the sequence for S1p1r (S1pr1-TKPTS). Cells were incubated with vehicle (saline) or cisplatin (20 mM) for 24 hours and then were labeled with various combinations of cell and mitochondrial markers. (A) FITC-phalloidin to label actin (green), MitoTracker (red), cytochrome

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Mitochondrial PCR Array Reveals Change in Expression of Genes Important in Mitochondrial Dynamics Weobservedthatalteredmitochondrialdynamicsinthepresence or absence of S1P1 is associated with the response of cells to cisplatin and susceptibility to apoptosis. We next used PCR array profiling to examine changes in mitochondrial genes that may contribute to the observed changes in mitochondrial dynamics and stability in a cellular environment of S1P1 deletion (primary wt/wt tubular cells isolated from PepckCreS1pr1 compared with fl/fl PepckCreS1pr1 mice) or stable S1P1 overexpression (pcDNA- versus S1pr1-TKPTS). As demonstrated in the heat map of the 84 mitochondrial gene probe set (Supplemental Figure 6), there was increased expression in mitochondrial genes in S1P1 overexpressing TKPTS cells and decreased expression in S1P1- deficient primary tubule cells (Table 4). Although additional experiments are needed to explore the functional relevance of the regulation of specificgenesbyS1pr1 expression, there was a marked change in expression of genes important for fusion and fission (Table 4), consistent with our data on mitochondrial Figure 6. Overexpression of S1P1 in TKPTS cells alters mito- stability in the current studies. chondrial fragmentation state. (A and B) Electron micrographs demonstrating differences in mitochondrial morphology between control (A; control pcDNA-transfected; more compact mitochondria, DISCUSSION arrowheads) and S1P1-overexpressing (B; more elongated mitochondria, arrows) TKPTS cells. (C and D) Mitochondrial morphology S1P1 activation is important for maintaining cell viability; in TKPTS cells transfected with scrambled sequence (C) is not dif- global deletion is embryonically lethal.30 We previously dem- m ferent from cells with siRNA knockdown of S1P1 (D). Bar, 0.5 M. onstrated that the protective effect of S1P1 agonists FTY720 or SEW2871 in IRI12 was mediated by activation of S1P1 ex- efficiency is an indicator of the relative flux through the ATP pressed on PT cells, independent of lymphopenia.13 Cisplatin synthase and proton leak pathways.29 Cisplatin caused a dose- use in cancer therapy is often limited by nephrotoxicity, spe- dependent decrease in whole cell mitochondrial function (total cifically,PTinjury.Here,wedemonstratedthatFTY720,a basal respiration and ATP-linked respiration), whereas S1pr1- drug that is approved for use in treating multiple sclerosis, TKPTS cells were less susceptible to the effects of cisplatin than protects kidneys from cisplatin-induced injury. Administra- control cells (Figure 7, A–D). This indicates that S1P1 overex- tion of FTY720 before cisplatin reduced immune cell infiltra- pression helps preserve the ability of mitochondria to synthesize tion, preserved endothelial integrity, reduced apoptosis, and ATP via ATP synthase after cisplatin treatment. This was further attenuated the increase in cytokine/chemokine expression in fl/fl reflected in the marked decrease in oxygen consumption in kidney. Conditional knockouts (PepckCreS1pr1 ) demon- cisplatin-treated control cells, to nearly undetectable levels, com- strated the protective phenotype of PT-S1P1 in cisplatin neph- pared with more modest decreases in the two parameters in rotoxicity; cisplatin-induced injury was greater in the absence S1pr1-TKPTS cells (Figure 7, C and D). The reverse was found of PT-S1P1 both in vivo and in TKPTS cells after siRNA knock- when comparing mitochondrial bioenergetics in primary TEC down. Furthermore, the protective effect of FTY720 required fl/fl wt/wt fl/fl cultures isolated from PepckCreS1pr1 and PepckCreS1pr1 PT-S1P1 as demonstrated both in vivo (PepckCreS1pr1 )and kidneys. The susceptibility to cisplatin effects on mitochondrial in vitro (siRNA knockdown of S1pr1 in TKPTS cells). The loss respiration was greater with PT-S1P1 deletion (therefore, only of FTY720 effectiveness in knockdown experiments, even lower cisplatin concentrations were used) than in control cells without 100% deletion, could be due to a threshold for recep- (Figure 7, E–H). tor number. The possibility that receptor downregulation

c (white). Overlay of red MitoTracker and white cytochrome c appears pink in merged images. (B) Membrane potential–sensitive red/green JC-1 is green in the cytoplasm and red within mitochondria with high membrane potential. (C) FITC-phalloidin (green) and MitoSox Red, which fluoresces more brightly with increasing mitochondrial superoxide. (D) FITC-phalloidin (green) and MitoTracker (red) showing mitochondrial morphology. (E) BAX labeling (white) appears white to pink upon translocation to and overlay with mitochondria (Mito- Tracker, red). Blue, nuclei stained with DAPI. (F) Mean fluorescence intensity of MitoSox Red in cells incubated with increasing concentrations of cisplatin for 24 hours and analyzed by flow cytometry. Flow histograms from a representative experiment are shown in Supplemental Figure 5. n=3 and the experiment was repeated two times. Bar, 20 mm in A, C, and E; 10 mm in B and D.

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Figure 7. Overexpression of S1P1 in TKPTS cells stabilizes mitochondrial function. Control (pcDNA-TKPTS) and S1P1-overexpressing (S1pr1-TKPTS) cells were incubated with vehicle (saline) or cisplatin (20, 40, or 60 mM) for 24 hours, and OCR was measured in a mitochondrial stress test by using Seahorse XF extracellular ﬂux technology. After measuring basal respiratory rate, cells were treated sequentially with the ATP synthase inhibitor oligomycin (0.75 mM), the mitochondrial uncoupler FCCP (0.75 then 1 mM), and the

916 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 908–925, 2015 www.jasn.org BASIC RESEARCH could account for the loss of FTY720 protection in these cells antitumor activity, FTY720 may itself be useful in treating will require future experiments examining receptor internali- cancer and could theoretically potentiate the antitumor effect zation, desensitization, functional uncoupling, or phosphory- of cisplatin. FTY720 can (1) induce G1 phase arrest and apo- lation state. We find in vivo that both homozygous knockouts ptosis leading to inhibition of gastric cancer cell prolifera- and mice heterozygous for PT S1pr1 deletion are more suscep- tion,35 (2) produce caspase-independent cell death of acute tible to injury, and FTY720 does not provide protection from lymphoblastic leukemia cells,36 and (3) mediate lung tumor IRI.13 Overexpression of S1pr1 rendered TKPTS cells resistant suppression via activation of PP2A-RIPK1–dependent necro- to cisplatin and increased mitochondrial stability, which may sis/apoptosis.37 contribute to the S1P1-mediated protective mechanism in Our analysis of mitochondrial structure and function was these cells. These studies, while further emphasizing the protec- prompted both by the importance of mitochondria as a target in tive role of S1P1 in AKI, demonstrate that by directly targeting cisplatin toxicity and the role of mitochondrial fragmentation in S1P1 on PTepithelial cells, FTY720 could provide a novel mech- cell injury in the kidney.35,38,39 PT cells do not rapidly divide; anism for protection from cisplatin-induced nephrotoxicity. therefore, mechanisms other than nuclear DNA cross-linking In AKI,9,31,32 inflammation plays a key role in cisplatin neph- may be important in cisplatin nephrotoxicity. Accumulation of rotoxicity.8 Although TNF-a contributes prominently to cis- positively charged cisplatin metabolites inside negatively charged platin nephrotoxicity by inducing cytokines/chemokines and mitochondria may render mitochondrial DNA more susceptible increasing immune cell infiltration, the role of other mediators to cross-linking than nuclear DNA. Renal PTs may therefore be is not as well defined. We have shown that FTY720 has a direct especially sensitive to cisplatin because of their high density of effect on PT-S1P1 to protect kidneys from the cytotoxic effects of mitochondria and the potential for activation of apoptosis and cisplatin. Any potential protective effect of lymphopenia on necrosis through a variety of pathways, including the mitochon- AKI33 may have been masked by the absence of PT-S1P1, as drial pathway. The dynamic balance of mitochondrial fission/ also seen in FTY720 protection from IRI.13 fusion in normal physiologic states preserves mitochondrial and Our in vitro studies with short-term siRNA knockdown stud- cellular homeostasis. These mitochondrial shape changes occur ies confirmed that the protective effect of FTY720 in vivo re- in healthy cells during processes such as cell division, to maintain quired PT-S1P1. In addition, our in vitro studies demonstrate mitochondrial number and function, but also in cell injury and that FTY720 acts directly on TKTPS cells without involvement of death, when they are accompanied by alterations in mitochon- immune cells8 to protect PT cells from direct toxicity. FTY720 drial respiration and by oxidative damage due to increased mi- could have an immunosuppressant effect in vitro,absentofim- tochondrial reactive oxygen species. Disruption of either fission mune cells, by reducing expression and release of proinflamma- or fusion can contribute to mitochondrial dysfunction.40 Hence, tory mediators from TKPTS cells (Table 3). In contrast with our there has been considerable interest in targeting mitochondria to receptor deletion experiments, overexpressing S1P1 in PT cells improve or maintain mitochondrial stability and prevent tissue enhances cell viability by reduced sensitivity to cisplatin toxicity, damage.41 suggesting that protection may derive from increased S1P1 sig- Our findings on mitochondrial respiratory function and naling driven by endogenous S1P and in an experimental ther- ultrastructure in S1P1-overexpressing TKPTS cells suggest that apeutic setting by FTY720. In experimental autoimmune S1P1 could be a suitable target for maintaining mitochondrial encephalomyelitis, a mouse model of multiple sclerosis, integrity in kidney tubule cells. Although they not conclusive, the FTY720 may have a direct neuroprotective effect that is depen- morphometric findings that mitochondria are longer and dent on the S1pr1 expression in astrocytes.34 In our studies, thinner in S1P1 overexpressing TKPTS cells suggest that the stimulation of PT-S1P1 reduced cisplatin-induced apoptosis, dynamic balance of fission and fusion may have shifted. This is perhaps by blocking the mitochondrial apoptotic pathway, but consistent with a protective phenotype, as similar ultrastructural additional studies are needed to further elucidate the mecha- analyses show that mitochondria are elongated in PTs of control nisms of PT-S1P1 stimulation. mice but are fragmented after IRI26; mitochondrial fragmenta- A relatively large number of effective experimental treat- tion contributes to disruption of mitochondrial function and ments have been identified for prevention of cisplatin neph- eventually cell death in AKI.42 The observed increases in mito- rotoxicity, but none have yet seen clinical use. Although our chondrial gene expression, such as those important for fission studies did not investigate whether FTY720 altered cisplatin’s and fusion, and in basal mitochondrial respiration rate in cells

complex I and III inhibitors antimycin A (10 mM) plus rotenone (1 mM) as indicated. (A) OCR for control (control pcDNA3.1 vector-transfected; pcDNA-TKPTS) and S1pr1-overexpressing TKPTS (S1pr1-TKPTS) cells treated with vehicle (Veh) or cisplatin (Cis). (B) OCR expressed as a percentage of total basal respiration for vehicle-treated control cells and S1pr1-overexpressing TKPTS cells treated with vehicle or cisplatin. Cisplatin-treated control cells have extremely low OCR and are not shown in B. (C and D) Respiratory control ratio (C) and coupling efficiency (D) for vehicle- and cisplatin-treated control and S1pr1-overexpressing cells. (E–H) Same experiments as A–D except with primary TEC cultures from kidneys of PepckCreS1pr1wt/wt and PepckCreS1pr1fl/fl mice and with incubations of only the lower concentrations (20 or 40 mM) of cisplatin. n=3. Values are the mean6SEM. ND, not detectable.

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Table 4. Identiﬁcation by PCR array proﬁling of mitochondrial genes that are regulated by deletion or overexpression of S1pr1

fl/fl S1pr1-TKPTS PepckCre-S1pr1 Symbol Description Related Function versus control versus control COX18 COX18 cytochrome c oxidase assembly Mitochondrial fission 4a 213.66b homolog and fusion FIS1 Fission 1 (mitochondrial outer membrane) 5.03a 29.01b homolog MFN1 Mitofusin 1 6.36a 27.37b DNM1L Dynamin 1-like 5.06a 210.79b MFN2 Mitofusin 2 4.32a 212.92b OPA1 Optic atrophy 1 (autosomal dominant) 8.75a 3.31a TIMM17B Translocase of inner mitochondrial Inner membrane 5.39a 4.65a membrane 17, homolog B translocation FXC1 (TIMM10b) Fracture callus 1 homolog 3.14a 6.44a IMMP2L IMP2 inner mitochondrial membrane 1.87 1.07 peptidase-like TAZ Tafazzin 3.32a 4.43a TIMM50 Translocase of inner mitochondrial 3.32a 7.88a membrane 50, homolog TIMM44 Translocase of inner mitochondrial 2.55a 4.11a membrane 44, homolog TIMM10 Translocase of inner mitochondrial 2.45a 210.35b membrane 10, homolog TOMM20 Translocase of outer mitochondrial Outer membrane 1.78 29.46b membrane 20 translocation TOMM22 Translocase of outer mitochondrial 3.1a 210.21b membrane 22 TOMM34 Translocase of outer mitochondrial 2.3a 29.27b membrane 34 TOMM40 Translocase of outer mitochondrial 1.75 3.34a membrane 40 TOMM40L Translocase of outer mitochondrial 2.69a 211.98b membrane 40L TOMM70a Translocase of outer mitochondrial 3.32a 29.46b membrane 70a AIP Aryl hydrocarbon receptor interacting Mitochondrion protein 4.5a 29.79b protein import COX10 COX10 homolog, cytochrome c oxidase 5.1a 27.58b assembly protein COX18 COX18 homolog, cytochrome c oxidase 4a 213.66b assembly protein DNAJC19 DnaJ (Hsp40) homolog, subfamily C, 4.38a 29.79b member 19 GRPEL1 GrpE-like 1 3.66a 9.7a HSPD1 Heat shock 60 kD protein 1 (chaperonin) 3.18a 14.5a MIPEP Mitochondrial intermediate peptidase 5.31a 27.02b SH3GLB1 SH3-domain GRB2-like endophilin B1 3.34a 18.74a AIP Aryl hydrocarbon receptor interacting Targeting proteins to 4.5a 29.79b protein mitochondria DNAJC19 DnaJ (Hsp40) homolog, subfamily C, 4.38a 29.79b member 19 FXC1(TIMM10B) Fracture callus 1 homolog 3.14a 6.44a GRPEL1 GrpE-like 1 3.66a 9.7a HSPD1 Heat shock 60 kD protein 1 (chaperonin) 3.18a 14.5a MIPEP Mitochondrial intermediate peptidase 5.31a 27.02b IMMP2L IMP2 inner mitochondrial membrane 1.87 1.07 peptidase-like

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Table 4. Continued

ﬂ/ﬂ S1pr1-TKPTS PepckCre-S1pr1 Symbol Description Related Function versus control versus control TSPO Translocator protein (18 kD) 5.78a 29.46b Slc25a1 Solute carrier family 25 (mitochondrial carrier; Small molecule 7.57a 26.93b citrate transporter), member 1 transport Slc25a10 Solute carrier family 25 (mitochondrial carrier; 4.69a 211.49b citrate transporter), member 10 Slc25a12 Solute carrier family 25 (mitochondrial carrier; 6.32a 212.48b citrate transporter), member 12 Slc25a13 Solute carrier family 25 (mitochondrial carrier; 9.92a 27.32b citrate transporter), member 13 Slc25a14 Solute carrier family 25 (mitochondrial carrier; 4.32a 213.66b citrate transporter), member 14 Slc25a15 Solute carrier family 25 (mitochondrial carrier; 4.82a 1.27 citrate transporter), member 15 Slc25a16 Solute carrier family 25 (mitochondrial carrier; 8.17a 1.04 citrate transporter), member 16 Slc25a17 Solute carrier family 25 (mitochondrial carrier; 3.76a 7.77a citrate transporter), member 17 Slc25a19 Solute carrier family 25 (mitochondrial carrier; 3.76a 21.6 citrate transporter), member 19 Slc25a2 Solute carrier family 25 (mitochondrial carrier; 5.06a 211.49b citrate transporter), member 2 Slc25a20 Solute carrier family 25 (mitochondrial carrier; 3.97a 2.6a citrate transporter), member 20 Slc25a21 Solute carrier family 25 (mitochondrial carrier; 1.06 211.49b citrate transporter), member 21 Slc25a22 Solute carrier family 25 (mitochondrial carrier; 3.48a 215.05b citrate transporter), member 22 Slc25a23 Solute carrier family 25 (mitochondrial carrier; 23.43a 27.37b citrate transporter), member 23 Slc25a24 Solute carrier family 25 (mitochondrial carrier; 2.62a 29.33b citrate transporter), member 24 Slc25a25 Solute carrier family 25 (mitochondrial carrier; 3.58a 4.11a citrate transporter), member 25 Slc25a27 Solute carrier family 25 (mitochondrial carrier; 3.76a 212.4b citrate transporter), member 27 Slc25a3 Solute carrier family 25 (mitochondrial carrier; 3.81a 80.9a citrate transporter), member 3 Slc25a30 Solute carrier family 25 (mitochondrial carrier; 5.39a 27.17b citrate transporter), member 30 Slc25a31 Solute carrier family 25 (mitochondrial carrier; 1 211.49b citrate transporter), member 31 Slc25a37 Solute carrier family 25 (mitochondrial carrier; 3.2a 211.65b citrate transporter), member 37 Slc25a4 Solute carrier family 25 (mitochondrial carrier; 2.91a 32.4 citrate transporter), member 4 Slc25a5 Solute carrier family 25 (mitochondrial carrier; 3.43a 32.4 citrate transporter), member 5 AIP Aryl hydrocarbon receptor interacting protein Mitochondrial 4.5a 29.79b transport BAK1 BCL2-antagonist/killer 1 6.11a 7.1a BCL2 B cell CLL/lymphoma 2 1.32 213.01b BCL2L1 BCL2-like 1 1.79 23.61b FXC1 Fracture callus 1 homolog (rat) 3.14a 6.44a

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Table 4. Continued

fl/fl S1pr1-TKPTS PepckCre-S1pr1 Symbol Description Related Function versus control versus control HSP90AA1 Heat shock protein 90 kD a (cytosolic), 1.89 28.95b class A member 1 HSPD1 Heat shock 60 kD protein 1 (chaperonin) 3.18a 14.5a IMMP2L IMP2 inner mitochondrial membrane 1.87 1.07 peptidase-like MFN2 Mitofusin 2 4.32a 212.92b MIPEP Mitochondrial intermediate peptidase 5.31a 27.02b MTX2 Metaxin 2 3.58a 29.93b STARD3 StAR-related lipid transfer (START) domain 4.38a 1.75 containing 3 TRP53 Tumor protein p53 3.97a 210b TSPO Translocator protein (18 kD) 5.78a 29.46b UCP1 Uncoupling protein 1 (mitochondrial, 1 211.49b proton carrier) UCP2 Uncoupling protein 2 (mitochondrial, 4.08a 41.87a proton carrier) UCP3 Uncoupling protein 3 (mitochondrial, 5.78a 211.49b proton carrier) BAK1 BCL2-antagonist/killer 1 Membrane polarization 6.11a 7.1a and potential BCL2 B cell CLL/lymphoma 2 1.32 213.01b BCL2L1 BCL2-like 1 1.79 23.61b TRP53 Tumor protein p53 3.97a 210b SOD1 SOD 1, soluble 6.02a 29.27b BNIP3 BCL2/adenovirus E1B interacting protein 3 14.93a 22.77b Pathway expression analysis of genes sorted and grouped by related mitochondrial functions. DC(t) was calculated for each gene of interest followed by the DDC(t) DD between groups and then fold change in expression (22 Ct) relative to the appropriate control, shown here as upregulation (positive values for fold change) or downregulation (negative values for fold change). Only absolute values for fold change .2arenoted. aPositive value for fold change. bNegative value for fold change. overexpressing S1P1 support the concept that S1P1 play a role in CONCISE METHODS mitochondrial stabilization. In addition, increased PGC-1a expression in these cells (data not shown) is consistent with in- Materials creased mitochondrial biogenesis and the removal of reactive Cisplatin (Sigma-Aldrich, St. Louis, MO), requiring reconstitution in oxygen species to preserve cell function.43 Conversely, increased saline, was used for initial experiments; subsequently, a formulation that susceptibility to injury in mice or cells deficient in S1P1 is con- is available in solution (1 mg/ml; Teva Parenteral Medicines Inc., Irvine, sistent with the decreases in gene expression revealed in the CA)wasused.Bothformulationsproducedthesameexperimentaleffect. mitochondrial array and with observed changes in indicators FTY720 and FTY720-p, the phosphorylated, active form of FTY720, of disrupted mitochondrial health (increased apoptosis and cy- werekindly providedbyNovartis(Basel,Switzerland).Incontrastwithin tochrome c, changes in mitochondrial membrane potential, vivo studies, where the prodrug, FTY720, is activated through phos- BAX translocation). In contrast with stable S1P1-overexpressing phorylation by endogenous sphingosine kinase 2, the phosphorylated, cells, mitochondrial morphometrics did not change in the tran- active form of FTY720, FTY720-p, was used for in vitro studies. sient knockdown experiments, but response to this short-term change in receptor expression may only be revealed after injury. Animals and Drug Administration It remains to be determined whether the protective effect of S1P All experiments were performed in accordance with the National on mitochondria is mediated through intracellular cascades trig- Institutes of Health (NIH) Guide for the Care and Use of Laboratory gered by binding to its cell surface receptor (S1P1) or through a Animals, and all procedures were approved by the University of Virginia direct effect of S1P on mitochondria.44,45 Animal Care and Use Committee. Experiments were performed on 10- In summary, we have demonstrated that PT-S1P1 is an to12-week-oldmaleC57Bl/6mice;WTmiceforinitialexperimentswere important target to attenuate cisplatin-induced AKI by pre- from the National Cancer Institute (Frederick, MD). Global S1pr1 de- fl/fl serving mitochondrial function and morphology. Weconclude ficiency is embryonically lethal.30 PepckCreS1P1 mice were generated fl/fl that targeting PT-S1P1 may represent a novel strategy in the by breeding S1pr1 (generously provided by Dr. Richard L. Proia, prevention of cisplatin-induced AKI. NIH) and PepckCre mice (a kind gift of Dr. Volker Haase, Vanderbilt

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University)46 as previously described13 and littermates with WT S1pr1 cisplatin or FTY720-p were the same for untransfected cells, and cells alleles were used as controls. Mice were maintained on a standard diet were harvested after 24 hours. These concentrations and periods of and water was freely available. Cisplatin was dissolved in saline at a incubation were selected from pilot experiments measuring cleaved- concentration of 1 mg/ml. Mice were given a single intraperitoneal in- caspase 3 by Western blot, as an indicator of apoptosis, in control TKPTS jection of either vehicle (saline) or cisplatin (27 mg/kg body wt). cells treated with varying concentrations of cisplatin (0–100 mm) with FTY720 was prepared in a 3% fatty acid–free BSA (Sigma-Aldrich)/ and without FTY720-p (10 nM) for varying periods of time (1–24 13PBS solution (BSA/PBS vehicle). FTY720 (240 mg/kg) or BSA/PBS hours). For electron microscopy, TKPTS cells were either transfected vehicle was administered intraperitoneally 1 hour before cisplatin and with siRNAs for S1p1r or scrambled sequence as above, or control cells again 1 and 2 days after cisplatin. Blood was collected for measurement were treated every day for 3 days with vehicle or FTY720 (10 nM) and of plasma creatinine, anticoagulated blood was analyzed for leukocyte harvested 24 hours after the last treatment. counts (HEMAVET 850; CDC Technologies, Oxford, CT), and mice were euthanized 72 hours after cisplatin. Isolation and Primary Culture of Renal TECs from wt/wt fl/fl PepckCreS1P1 and PepckCreS1P1 Mice Assessment of Kidney Function and Histology Primary TECs were isolated and propagated as described50 but with Plasma creatinine, as a measure of kidney function, was determined minor modifications. Briefly, kidneys were removed from 12- to 16- using a colorimetric assay according to the manufacturer’s protocol week-old male mice and placed in HBSS (Gibco, Carlsbad, CA) at 4°C, (Sigma-Aldrich). For histology, kidneys were fixed overnight in 0.2% with penicillin and streptomycin (13; Gibco). The capsule was removed sodium periodate/1.4% DL-lysine/4% paraformaldehyde in 0.1 M phos- and the cortex was trimmed away from the medulla using small curved phate buffer, pH 7.4 (4% PLP), and embedded in paraffin. Kidney scissors. Cortical pieces were minced with a razor blade and suspended sections were prepared for hematoxylin and eosin staining as previously in 10 ml HBSS containing collagenase type II (200 U/ml; Worthington described47 and viewed by light microscopy (Carl Zeiss AxioSkop) un- Chemical, Lakewood NJ) and an equal weight (relative to collagenase) of der 3200 magnification. Photographs were taken and brightness/ soybean trypsin inhibitor (Invitrogen) then placed on a rotator contrast adjustment was made with a SPOT RT camera (software ver- (70 rpm) in a 37°C incubator for 30 minutes. The suspension was then sion 3.3; Spot Imaging Solutions/Diagnostic Instruments Inc., Sterling split into two 15-ml conical tubes and 5 ml heat-inactivated horse serum Heights, MI). The histologic change in the outer medulla was expressed (Gibco) was added to each tube. The tubes were vortexed for 30 seconds as ATN, scored on a scale of 1–5, as previously described.12 and then left to sit upright, undisturbed for 1 minute. The top 7–8ml was transferred to a 50-ml conical tube. An aliquot of the tubule sus- Quantitative Real-Time RT-PCR pension was used to determine the total weight of the tubules in each Total RNA was extracted, and single-stranded cDNA was synthesized sample; the remaining tubules were pelleted by centrifugation at 200 as previously described.12,13 Primers were obtained from Integrated relative centrifugal force for 7 minutes at 4°C and washed once in HBSS. DNA Technologies (Coralville, IA); primer sequences for S1P1–5,12 The final tubule pellet was resuspended at 1 mg/ml in kidney culture CXCL1, IL-6, and TNF-a,28 and IL-1b and MCP-148 were as de- medium: DMEM/F-12 culture media (Gibco) with insulin, transferrin, scribed previously. Quantitative PCR was performed using a MyIQ and selenium (5 mg/ml, 2.75 mg/ml, and 3.35 ng/ml, respectively; Single Color Real-Time PCR Detection System iCycler (Bio-Rad). Gibco), hydrocortisone (40 ng/ml; Sigma-Aldrich), mouse EGF Samples were calculated with normalization to glyceraldehyde-3- (25 ng/ml; BD Biosciences, San Jose, CA), and penicillin and streptomycin phosphate dehydrogenase.13 (Gibco). Tubules were plated in 24-well (1.5 ml), 12-well (2.25 ml), and 6-well (3 ml) tissue culture plates for experiments. Media were changed Cell Culture: TKPTS Cells 24 hours after plating and every 48–72 hours thereafter. EGF was only Mouse PT kidney cells (TKPTS cells; E. Bello-Reuss, Texas Tech included in the media for the first 24 hours after plating. TECs migrate University, Galveston, TX), cells from a mouse PT cell line,49 were out of the tubules and form a confluent monolayer by days 7–10. The maintained in complete DMEM/F12 medium (Invitrogen, Carlsbad, epithelial origin of the TECs was confirmed by prominent E-cadherin CA; supplemented with 10% FBS and 1% penicillin/streptomycin) at immunoreactivity (Clone DECMA-1; eBioscience, San Diego, CA; Sup-

37°C in a humidified 5% CO2 atmosphere. For siRNA experiments, plemental Figure 7). Cells were used between days 7 and 10 of culture. In TKPTS cells were seeded in 12-well plates in complete medium without addition, genotyping of samples of tail and isolated tubules from wt/wt fl/fl antibiotics the day before the experiment. Two ready-to-use validated PepckCreS1P1 and PepckCreS1P1 mice using PCR and primers double-stranded 21-nucleotide siRNAs for S1p1r (D-051684-01 and selective for the wt and floxed alleles (as previously described13) con- fl/fl D-051684-04; 25 nM) were transfected into TKPTS cells along with firmed our prior characterization of the PepckCreS1P1 mice13 and ON-TARGETplus Nontargeting siRNA (D-002810-01; Dharmacon demonstrated conclusively for the first time that S1pr1 is deleted from Inc., Lafayette, CA) using oligofectamine (Invitrogen) diluted in Opti- kidney tubules (Supplemental Figure 7C). MEM I (Invitrogen) overnight according to the manufacturer’sinstruc- tions. Transfected cells were then incubated with fresh complete Stable Overexpression of S1pr1 in TKPTS Cells DMEM/F12 medium for a total period of 48 hours before RNA extrac- Confluent monolayers of TKPTS cells were transfected either with tion or before treatment with cisplatin (20 mM; 24 hours) or FTY720-p pcDNA3.1/V5-His expression vector encoding the mouse S1P1 (10 nM; 1 hour before cisplatin). The phosphorylated, active form of (S1pr1-TKPTS) or the control pcDNA3.1 vector (pcDNA-TKPTS) FTY720, FTY720-p, is needed for in vitro studies. Incubations with using Lipofectamine 2000 (Invitrogen) according to the manufac-

J Am Soc Nephrol 26: 908–925, 2015 Proximal Tubule S1P1 in Mitochondrial Function 921 BASIC RESEARCH www.jasn.org turer’s instructions. Cells were grown for 24 hours and washed with FACS Analyses PBS before treatment with geneticin (G418 sulfate; 300–500 mg/ml; Flow cytometry was used to analyze kidney leukocyte content 72 hours Gibco) in DMEM/F12. Individual colonies, obtained at 2–3weeks, after cisplatin. Kidneys were extracted, minced, and digested as were expanded. Stably transfected pcDNA-TKPTS and S1pr1-TKPTS previously described.53 After blocking nonspecific Fc binding with were maintained in complete medium supplemented with G418 for anti-mouse CD16/32 (2.4G2), fresh kidney suspensions were incu- selection of cells with neomycin resistance. The level of expression (by bated with fluorophore-labeled anti-mouse CD45 (30-F11). CD45- RT-PCR) of S1P1 in S1pr1-TKPTS was 29-fold compared with labeled samples were then incubated with different combinations of pcDNA-TKPTS-transfected cells (Supplemental Figure 5, A and B), fluorophore-labeled mouse anti-F4/80 (BM8), GR-1 (Ly6G), or and there was no change in expression of S1P3–S1P5 or sphingosine CD11b. 7-AAD (BD Biosciences) was added 15 minutes before ana- kinases 1 and 2 (data not shown). Western blot showed a band of the lyzing the samples to separate live from dead cells. Subsequent flow correct predicted molecular weight for S1P1 (rabbit3S1P1, 1:250, cytometry data acquisition was performed on FACS Calibur (Becton clone H-60; Santa Cruz Biotechnology, Dallas, TX51,52)incytoplas- Dickinson, San Jose, CA) with Cytek 8 color flow cytometry upgrade mic and membrane fractions of cell lysates of S1pr1-TKPTS that was (Cytek Development, Inc., Fremont, CA). Data were analyzed by not visible in the membrane fraction after treatment of cells with FlowJo software 9.0 (Treestar, Ashland, OR). Unless otherwise noted, 10 nM FTY720-p for 24 hours (presumably due to internalization all antibodies were purchased from eBioscience and were used at a of the receptor). S1P1 was undetectable in untransfected TKPTS cells concentration of 5 mg/ml. or pcDNA-TKPTS cells (Supplemental Figure 5C). Cell Viability Assay Western Blot Analyses Viable cells in TKPTS cell or primary TEC cultures were measured by Cell lysates were prepared from TKPTS cells or primary TECs by using a colorimetric assay that is based on the detection of de- sonicating in RIPA lysis buffer (Thermo Fisher Scientific) enriched with hydrogenase activity (Cell Counting Kit-8; Dojindo Molecular 1% protease and phosphatase inhibitor cocktail (formulation: sodium Technologies Inc.). Cells were cultured in 96-well plates (25–30,000 fluoride, sodium orthovanadate, b-glycerophosphate, sodium pyro- cells in a volume of 100 ml media) the day before experiments. Cells phosphate, aprotinin, bestatin, E64, leupeptin, EDTA; Thermo Fisher were incubated with vehicle or cisplatin (20 mM) for 20 hours; 2 Scientific). Kidney samples were prepared in the same buffer but tissues hours before the end of the incubation, WST-8, the kit’s tetrazolium 2 were first homogenized by shaking with metal beads (50 s 1,10min- salt, was added to wells according to the manufacturer’s directions, utes) using a TissueLyser (Qiagen) followed by sonication to improve and cultures were protected from light. The resulting orange-colored protein extraction. Homogenates were centrifuged (12,000 rpm, 15 min- formazan, formed by reduction of WST-8, was detected by measuring utes, 4°C), and the protein concentration in the supernatant was mea- absorbance at 450 nm, and OD was corrected for background absor- sured by using the BCA protein assay kit (Pierce). Equal volumes of the bance of media alone. remaining cell or tissue lysate supernatants were boiled with Leammli buffer and b-mercaptoethanol (10 minutes, 100°C); proteins were sep- Immunofluorescence Labeling and Microscopy arated using 15% SDS-PAGE and transferred to polyvinylidene fluoride TKPTScellsandprimarytubuleepithelialcellsweregrownonglassslides membranes (EMD Millipore). The membranes were blocked with Od- with flexiPERM silicon chambers (Sarstedt, Newton, NC). TKPTS cells yssey blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 hour at and TECs were fixed with 4% paraformaldehyde in 0.1 M phosphate room temperature and then incubated with primary antibody (mouse buffer, pH 7.4, for 30 minutes. Kidneys were fixed in 1% PLP (1% anti–b-tubulin, 1:5000, 3F3-G2; Santa Cruz Biotechnology), anti-NaK- paraformaldehyde, 1.4% DL-lysine, 0.2% sodium periodate in 0.1 M ATPase (1:500; Abcam, Inc. Cambridge, MA) or anti–glyceraldehyde-3- phosphate buffer, pH 7.4) overnight, incubated in 30% sucrose for 48 phosphate dehydrogenase (6C5, 1:10,000; Ambion, Austin, TX) to hours at 4°C, and embedded and frozen in optimal cutting temperature compensate for variations in protein loading, rabbit anti–caspase-3 (rec- compound (Ted Pella Inc.). Frozen sections (7 mm) or fixed cells were ognizes both full-length and cleaved-caspase 3, 1:1000; Cell Signaling permeabilized with 0.3% Triton X-100, and nonspecific binding was Technology, Boston, MA), or rabbit anti-BAX (1:500; Cell Signaling blocked with 10% horse serum (cells and kidney sections) and anti- Technology) diluted in Odyssey blocking buffer/0.1% Tween. The mem- mouse CD16/32 (10 mg/ml, kidney sections only; clone 2.4G2; StemCell branes were washed twice for 5 minutes each in 0.05% Tween/phosphate Technologies Inc, Vancouver, BC, Canada). Sections or cells were la- buffer solution, probed with goat anti-rabbit IRDye 680RD- and goat beled with FITC-labeled anti-neutrophil mAb (7 mg/ml, clone 7/4; anti-mouse IRDye 800CW-labeled secondary antibody (1:20,000; Cedarlane, Burlington, NC), APC-labeled rat anti-CD31/PECAM-1 LI-COR) in Odyssey blocking buffer/0.1% Tween/0.01% SDS for 1 (5 mg/ml, clone MEC 13.3; BioLegend, San Diego, CA), or APC-labeled hour at room temperature, and washed twice for 5 minutes in Tween/ rat anti-F4/80 (7 mg/ml, clone BM8; Invitrogen) for 1 hour. Kidney phosphate buffer solution. Air-dried membranes were imaged and sections or cells were stained with rabbit anti-mouse CXCL1 or MCP- densitometric analysis of bands was performed by using a LI-COR 1(2.5mg/ml; PeproTech, Rocky Hill, NJ) followed by Cy3-labeled don- Odyssey Infrared Imaging System with Odyssey 3.0 analytical software key anti-rabbit IgG (1:1000; Jackson ImmunoResearch Laboratories, (LI-COR) for multiplex detection of the two different antigens. West Grove, PA). Apoptotic cells were detected by TUNEL assay (In In some experiments, mitochondrial and membrane fractions of Situ Cell Death Detection kit, TMR red; Roche) according to the man- TKPTS cells were prepared using a commercial kit (Thermo Fisher ufacturer’s instructions. For mitochondrial studies, TKPTS cells or pri- Scientific), and 20 mg of protein from each fraction was loaded per lane. mary TECs were labeled with mouse anti-BAX (1:100 [Cell Signaling

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Technology]; followed by Alexa647-labeled donkey anti-mouse IgG, TKPTS cells were seeded in Seahorse 24-well tissue culture plates at a 1:500 [Molecular Probes]), MitoTrackerRed (25 nM, 30 minutes at density of 2.53104 cells/well and allowed to adhere for 24 hours. 37°C; Invitrogen), mouse anti-cytochrome c (1:100 [Cell Signaling Primary cultures of TECs from kidney were grown to confluence on Technology]; followed by Alexa647-labeled donkey anti-mouse IgG, Seahorse 24-well tissue culture plates, as described above. Cells were 1:500 [Molecular Probes]), MitoSox (5 mM, 10 minutes; Invitrogen), treated with vehicle (saline) or cisplatin (20–60 mM for TKPTS cells; or JC-1 (10 mg/ml, 20 minutes at 37°C; Invitrogen). JC-1 is a mitochon- 20–40 mM for primary tubule cells) for 20 hours. Before the assay, the drial membrane potential–sensitive carbocyanine probe; monomeric media was changed to unbuffered DMEM (12800-017, pH 7.4, 37°C; green fluorescent JC-1 is taken up into healthy higher membrane po- Gibco), and cells were equilibrated for 30 minutes at 37°C. After mea- tential mitochondria, where it reversibly forms red-fluorescent aggre- suring basal respiratory rate, oligomycin (uncouples ATP-coupled res- gates, whereas with loss of mitochondrial membrane potential, JC-1 piration by inhibiting ATP synthase, 5 mM; Sigma-Aldrich), FCCP remains in the cytoplasm or diffuses out of mitochondria and is green. (750 nM and 1 mM, mitochondrial uncoupling agent that uncouples Actin was labeled with phalloidin-FITC (1 mg/ml; Sigma-Aldrich). Pri- mitochondrial respiration from ATP and reveals maximal respiratory mary TECs were labeled with E-cadherin (see Supplemental Figure 7 for rate; Sigma-Aldrich), and rotenone (1 mM; Sigma-Aldrich) with anti- details). Nuclei were visualized using 49,6-diamidino-2-phenylindole mycin A (electron transport chain [complex I and III) inhibitors, (DAPI). All specimens were mounted with ProLong Gold Antifade re- eliminates all mitochondrial respiration, 10 mM; Sigma-Aldrich) agent with DAPI (Invitrogen). Images were acquired using the Carl Zeiss were injected sequentially during the assay. FCCP was used at two Axiovert 200 microscopy system with ApoTome imaging and Axiovision doses and monitored over time because it has a narrow dosing range software (Carl Zeiss Microscopy, LLC, Thornwood, NY). in which it induces the maximal rate of mitochondrial respiration.54 OCR was measured in 3-minute periods of time (over a total period of Electron Microscopy 2 hours). Basal mitochondrial respiration, ATP-linked respiration, Confluent cultures of TKPTS cells (stably expressing empty pcDNA proton leak (non-ATP–linked oxygen consumption), maximal respi- vectorormS1P1construct, or after transientS1pr1siRNAknockdown ration, nonmitochondrial respiration, reserve respiratory capacity, re- or FTY720 treatment) were harvested, fixed with 2% paraformalde- spiratory control ratio, and coupling efficiency were determined in hyde/2% glutaraldehyde in 0.1 M phosphate buffer for 45 minutes, whole cells according to Brand et al.29 Three to four wells were used for washed with cold PBS and prepared for electron microscopy by each experimental group. incubating in 2% osmium (in 0.1 M phosphate buffer, pH 7.4) for 1 Parameters of mitochondrial function were calculated from the hour and then washing and dehydrating through a graded series of respiration rates measured at times before and after the addition of ethanol, and embedding in EmBed812 embedding resin (Electron modulators of mitochondrial function. Basal mitochondrial respira- Microscopy Sciences, Fort Washington, PA). Serial ultrathin sections tion is total basal respiration rate minus nonmitochondrial respira- (80–90 nm) of TKPTS cells were collected on copper mesh grids and tion. ATP-linked respiration is revealed by the degree of decrease from stained with uranyl acetate and Reynolds lead citrate. Two to three basal respiration in the presence of oligomycin. Maximal mitochon- grids from each sample were examined using a JEOL 1230 transmis- drial respiration is measured after the addition of FCCP (with sion electron microscope, and digital photographs of cells were cap- subtraction of nonmitochondrial respiration). Mitochondrial reserve tured by real-time digital imaging. capacity is the maximal minus basal mitochondrial respiration. Proton leak is the oligomycin-induced respiration minus nonmito- Ultrastructural Analyses chondrial respiration. Finally, nonmitochondrial respiration is the Electron micrographs were opened in StereoInvestigator software (MBF oxygen consumption remaining when the mitochondrial electron Bioscience,Williston, VT), whichwas calibratedtomatch the scale ofthe transport chain is inhibited by rotenone and antimycin A. images, and mitochondrial contours within 20–25 cells/group (yielding .300 mitochondrial contours/group) were drawn to obtain an unbi- Mitochondrial PCR Array Profiling ased estimate of mitochondrial morphology. Contour measurements Total RNA from TKPTS (pcDNA- and S1pr1-transfected; three (feret minimum and maximum, area, perimeter, shape factor, compact- culture plates of each, pooled) and primary epithelial cells (from wt/wt fl/fl ness, roundness, and convexity) were automatically calculated using PepckCreS1pr1 and PepckCreS1pr1 mice; cultures from three embedded equations within the software. Feret maximum (the longest mice each, pooled) was extracted using TriReagent (Life Technolo- projection of the minimal bounding box, i.e., parallel tangents apposing gies, Grand Island, NY). Total RNA was treated with RNase-free opposite sides of the profile), compactness (values are 0–1; a circle has a DNase, and RT was performed with 1 mgoftotalRNAusinganRT2 value of 1), and shape factor (defines a relationship of perimeter to First-Strand Kit (SABioscience, Frederick, MD). Approximately 900 ng area; a circle has a value of 3.54) are reported. of cDNA was processed for quantitative real-time RT-PCR (RT- qPCR) of 84 genes involved in mitochondrial biogenesis and 12 2 Measurements of Oxygen Consumption Rate and housekeeping genes, including internal controls, by using an RT Pro- 2 Mitochondrial Function in TKPTS Cells and Primary filer PCR Array Kit (RT Profiler PCR Array Mouse Mitochondria, Cultures of Kidney PT Cells PAMM-087Z; SABioscience) and Bio-Rad CFX96 real-time PCR The oxygen consumption rate (OCR), as an indicator of mitochon- system. PCR products were quantified by measuring SYBR Green drial bioenergetics in whole cells, was measured using a Seahorse fluorescent dye, and threshold cycle (Ct) values were calculated. An XF-24FluxAnalyzer(SeahorseBiosciences,Billerica,MA)asdescribed.54 integrated web-based software package for the PCR Array System

J Am Soc Nephrol 26: 908–925, 2015 Proximal Tubule S1P1 in Mitochondrial Function 923 BASIC RESEARCH www.jasn.org performed DDCt-based fold-change calculations from the uploaded 8. Miller RP, Tadagavadi RK, Ramesh G, Reeves WB: Mechanisms of Cis- raw threshold cycle data. platin nephrotoxicity. Toxins (Basel) 2: 2490–2518, 2010 9. Bonventre JV, Yang L: Cellular pathophysiology of ischemic acute kidney injury. JClinInvest121: 4210–4221, 2011 Statistical Analyses 10. Li L, Okusa MD: Macrophages, dendritic cells, and kidney ischemia- GraphPad Instat 3 (GraphPad Software Inc., La Jolla, CA), SigmaPlot reperfusion injury. Semin Nephrol 30: 268–277, 2010 11.0 (Systat Software Inc., Chicago, IL), and Canvas X (ACD Systems 11. Iwata M, Herrington J, Zager RA: Sphingosine: A mediator of acute International Inc., Seattle, WA) were used to analyze and present the renal tubular injury and subsequent cytoresistance. Proc Natl Acad Sci – data. Data were analyzed, after transformation if needed to generate a USA92: 8970 8974, 1995 t 12. Awad AS, Ye H, Huang L, Li L, Foss FW Jr, Macdonald TL, Lynch KR, normal distribution, by two-tailed test or one- or two-way ANOVA Okusa MD: Selective sphingosine 1-phosphate 1 receptor activation with post hoc analysis as appropriate. P,0.05 was used to indicate reduces ischemia-reperfusion injury in mouse kidney. Am J Physiol significance. Renal Physiol 290: F1516–F1524, 2006 13. Bajwa A, Jo SK, Ye H, Huang L, Dondeti KR, Rosin DL, Haase VH, Macdonald TL, Lynch KR, Okusa MD: Activation of sphingosine-1- phosphate 1 receptor in the proximal tubule protects against ischemia- – ACKNOWLEDGMENTS reperfusion injury. JAmSocNephrol21: 955 965, 2010 14. Spiegel S, Milstien S: Functions of a new family of sphingosine-1- phosphate receptors. Biochim Biophys Acta 1484: 107–116, 2000 We gratefully acknowledge the University of Virginia Advanced 15. Ancellin N, Colmont C, Su J, Li Q, Mittereder N, Chae SS, Stefansson S, Microscopy Facility for ultrathin sectioning and staining and the Liau G, Hla T: Extracellular export of sphingosine kinase-1 enzyme. University of Virginia Research Histology core, Dr. V. Brinkmann Sphingosine 1-phosphate generation and the induction of angiogenic – (Novartis, Basel, Switzerland) for providing FTY720-p, Dr. Elsa Bello- vascular maturation. JBiolChem277: 6667 6675, 2002 16. Spiegel S, Milstien S: Sphingosine 1-phosphate, a key cell signaling Reuss (Texas Tech University) for providing TKPTS cells, and all molecule. J Biol Chem 277: 25851–25854, 2002 members of the Okusa laboratory. 17. 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