Brief Communications

Mitochonic Acid 5 Binds Mitochondria and Ameliorates Renal Tubular and Cardiac Myocyte Damage

Takehiro Suzuki, Hiroaki Yamaguchi, Motoi Kikusato, Osamu Hashizume, Satoru Nagatoishi, Akihiro Matsuo, Takeya Sato, Tai Kudo, Tetsuro Matsuhashi, Kazutaka Murayama, Yuki Ohba, Shun Watanabe, Shin-ichiro Kanno, Daichi Minaki, Daisuke Saigusa, Hiroko Shinbo, Nobuyoshi Mori, Akinori Yuri, Miyuki Yokoro, Eikan Mishima, Hisato Shima, Yasutoshi Akiyama, Yoichi Takeuchi, Koichi Kikuchi, Takafumi Toyohara, Chitose Suzuki, Takaharu Ichimura, Jun-ichi Anzai, Masahiro Kohzuki, Nariyasu Mano, Shigeo Kure, Teruyuki Yanagisawa, Yoshihisa Tomioka, Masaaki Toyomizu, Kohei Tsumoto, Kazuto Nakada, Joseph V. Bonventre, Sadayoshi Ito, Hitoshi Osaka, Ken-ichi Hayashi and Takaaki Abe
JASN July 2016, 27 (7) 1925-1932; DOI: https://doi.org/10.1681/ASN.2015060623

Abstract

Mitochondrial dysfunction causes increased oxidative stress and depletion of ATP, which are involved in the etiology of a variety of renal diseases, such as CKD, AKI, and steroid–resistant nephrotic syndrome. Antioxidant therapies are being investigated, but clinical outcomes have yet to be determined. Recently, we reported that a newly synthesized indole derivative, mitochonic acid 5 (MA-5), increases cellular ATP level and survival of fibroblasts from patients with mitochondrial disease. MA-5 modulates mitochondrial ATP synthesis independently of oxidative phosphorylation and the electron transport chain. Here, we further investigated the mechanism of action for MA-5. Administration of MA-5 to an ischemia-reperfusion injury model and a cisplatin–induced nephropathy model improved renal function. In in vitro bioenergetic studies, MA-5 facilitated ATP production and reduced the level of mitochondrial reactive oxygen species (ROS) without affecting activity of mitochondrial complexes I–IV. Additional assays revealed that MA-5 targets the mitochondrial protein mitofilin at the crista junction of the inner membrane. In Hep3B cells, overexpression of mitofilin increased the basal ATP level, and treatment with MA-5 amplified this effect. In a unique mitochondrial disease model (Mitomice with mitochondrial DNA deletion that mimics typical human mitochondrial disease phenotype), MA-5 improved the reduced cardiac and renal mitochondrial respiration and seemed to prolong survival, although statistical analysis of survival times could not be conducted. These results suggest that MA-5 functions in a manner differing from that of antioxidant therapy and could be a novel therapeutic drug for the treatment of cardiac and renal diseases associated with mitochondrial dysfunction.

Mitochondria are key organelles implicated in a variety of processes related to energy and free radical generation and various signal pathways.1 The kidney is a highly energetic organ that contains large amounts of mitochondria. Mitochondrial dysfunction plays a critical role in the pathogenesis of the common pathways of CKD and AKI.2 In addition, recently, it has been found that the genes responsible for steroid–resistant nephrotic syndrome (SRNS) are not only the components of slit diaphragm but that mitochondrial genes in the podocytes are also involved (e.g., ADCK4 and COQ6).3 Clinically antioxidant quinones (e.g., CoQ10 and idebenone) have been used to retard the disease progression of renal and neurorenal damage in patients with mitochondrial diseases.2 However, because the effectiveness of such quinones has not been well established,4 a more effective therapeutic approach is needed.

Here, we report that a newly synthesized indole acetic acid (IAA) derivative, mitochonic acid 5 (MA-5),5 targets the mitochondrial protein mitofilin and facilitates ATP production, protecting against AKI and renal respiration damage in Mitomice. Our finding changes the focus of the search for drugs for mitochondrial and mitochondrial–related renal diseases, such as AKI, CKD, and SRNS.

To examine the tissue-protective effect of MA-5, we initially examined the oral bioavailability.6 As in Figure 1A, oral administration of MA-5 increased the plasma concentration in a dose-response manner at the peak time of 1 hour. Then, we examined the tissue-protective effect of MA-5 using a bilateral ischemia-reperfusion injury (IRI) model.7 After IRI, the MA-5–treated group showed a significantly improved plasma creatinine level after reperfusion (P<0.05) (Figure 1B). In addition, representative AKI marker KIM-1 expression at the outer medullar was measured and shown to be significantly decreased in MA-5–treated IRI mice at 48 hours (Figure 1B, upper panel). Histologic examination also showed that the acute tubular necrosis score was significantly reduced in MA-5–treated mice kidneys (Figure 1B, lower panel). These data suggest that decreased tubular cell injury after ischemia surgery contributed to the improved kidney pathology and renal function.

Figure 1.
Figure 1.

MA-5 rescued the IRI kidney model, and the direct effects of MA-5 on mitochondria are shown. (A) Plasma concentration of MA-5 after oral administration at a dose of 25, 50, or 150 mg/kg (n=3). (B) Plasma creatinine (Cr), KIM-1 immunostaining–positive area, and acute tubular necrosis (ATN) score of kidney histology were significantly reduced in MA-5–treated mice 48 hours after IRI surgery (vehicle [Veh] sham, n=4; Veh IRI, n=5; MA-5 IRI, n=6). KIM-1 stain (×20 and ×100 magnification) and periodic acid–Schiff (PAS) stain (magnification ×200) of the kidney pathology 48 hours after sham or IRI surgery. *P<0.05; **P<0.01 (two-way ANOVA and Tukey post hoc test for plasma Cr, unpaired two–tailed t test for KIM-1 area, and ATN score versus Veh IRI). (C) Plasma BUN and ATN score of kidney histology were significantly reduced in the MA-5–treated mice 96 hours after cisplatin administration (vehicle [Veh] -CIN, n=7; MA-5-CIN, n=8). PAS stain (magnification ×200) of the kidney pathology 96 hours after intraperitoneal cisplatin injection. *P<0.05 (two-way ANOVA and Tukey post hoc test for plasma BUN and unpaired two–tailed t test for ATN score versus Veh-CIN). (D) ATP production of isolated detergent–solubilized rat hepatic mitochondria with or without MA-5 (10 μM; n=5). ADP (64 nM) was added to the reaction medium. (E) Respiration rate of mitochondria in states 2–4. (F) Mitochondrial ROS production in mouse cardiac mitochondria was measured with DMSO (0.1%) or MA-5 (10 μM; n=5). (G) Effect of MA-5 on the activities of complexes I–IV using detergent–solubilized bovine heart mitochondria (n=3 for complex I experiment; n=6 for complexes II–IV experiments). *P<0.05 (unpaired two–tailed t test versus DMSO). KCN, potassium cyanide; mOD, milli optical density; RLU, relative luciferase unit; TTFA, 2- thenoyltrifluoroacetone.

In addition, we accessed the MA-5 effect in a mouse cisplatin–induced nephropathy (CIN) model. The main part of cisplatin nephrotoxicity occurs in tubular epithelial cells, with tubular cell death through either necrosis or apoptosis caused by cisplatin toxicity. Morphologic and functional mitochondrial damages are key factors in renal tubular disruption in cisplatin nephropathy. In the mouse CIN model, plasma BUN was significantly decreased in the MA-5–treated mice at 96 hours after cisplatin administration (P<0.05) (Figure 1C). Acute tubular necrosis in the outer medullar area was also significantly reduced in the CIN mice group with MA-5 gavage compared with vehicle corn oil–administered CIN animals (Figure 1C).

Ischemia-reperfusion evokes ATP depletion in tubular cells and results in AKI, and the rapid recovery and maintenance of the ATP levels after reperfusion are crucial for minimizing the tissue damage and promoting the regeneration of the tubular epithelial cells. Recently, we have reported that MA-5 increased the cellular ATP level and the improved survival of fibroblasts from patients with mitochondrial diseases.5 Thus, MA-5 might protect tubular cells from IRI by cisplatin by maintaining intracellular ATP levels.

To examine the effect of MA-5 on mitochondria, we performed bioenergetic examinations. At first, we measured the ATP level of detergent-solubilized mitochondria, which cannot couple the ETC-dependent proton with ATP synthesis. As shown in Figure 1D, MA-5 significantly increased the mitochondrial ATP contents. We next analyzed the effect of MA-5 on the respiration state in mitochondria. In isolated cardiac mitochondria, no effects of MA-5 were detected on state 2 (proton leakage in the absence of ADP), state 3 (ATP synthesis), and state 4 (non-ATP synthesis) respiration (Figure 1E). Under this condition, mitochondrial reactive oxygen species (ROS) (Figure 1F) were reduced by MA-5. We further examined the effect of MA-5 on oxidative phosphorylation (OXPHOS) complexes I–IV activities in isolated mitochondria.8 No significant shifts in the presence of MA-5 were observed in complexes I–IV activities (Figure 1G). These data suggest that MA-5 facilitated mitochondrial ATP production and reduced mitochondrial ROS generation independently of OXPHOS/electron transport chain (ETC) as reported.5

Although the plant hormone auxin binds to its nuclear receptor TIR1 and its activation leads to transcriptional reprogramming,9 there is no known mammalian homolog, and, therefore, auxins are widely used as human-safe drugs for development or herbicides. On the basis of the above observations, we attempted to identify putative MA-5–binding protein(s) using ferrite glycidyl methacrylate (FG) beads10 conjugated with MA-5. The affinity beads were incubated with cell lysate, and bound proteins were separated by SDS-PAGE and subjected to tandem mass spectrometry (MS). The major affinity–purified proteins were identified as mitofilin, HSP70, ATP synthase subunits-α and -β, RuvBL1 and -2, VDAC1, and CHCHD3 (Figure 2A), all of which are, intriguingly, mitochondrial proteins with ATPase activity or binding. We next made a binding assay using radiolabeled IAA to identify the exact target molecule for MA-5. We used IAA for the competitive binding assay, because MA-5 is a synthetic derivative from IAA. Radiolabeled IAA absorbed on RuvBL1 and -2 and CHCHD3 protein was not released by cold IAA (Figure 2B, left panel). However, IAA binding to mitofilin was reversible (Figure 2B, right panel).

Figure 2.
Figure 2.

MA-5 binds mitochondrial inner membrane protein mitofilin. (A) Affinity purification of MA-5–binding proteins using magnetic FG beads conjugated with MA-5. (B) Binding of MA-5 to MINOS proteins. Effect of nonlabeled IAA (50 μM) on 25 nM 3H-IAA binding to RuvBL1 and -2 and CHCHD3 (left panel). Effect of nonlabeled IAA (50 μM) and MA-5 (50 μM) on 25 nM 3H-IAA binding to mitofilin (right panel). (C) LC/MS/MS–based Scatchard plot shows the binding of MA-5 to mitofilin (n=5). (D) The binding kinetics of MA-5 with Mitofilin d1–120 (recombinant mitofilin protein with a deleted transmembrane domain at the N terminus of mitofilin; 121–758 amino acids) measured by SPR. (E) MA-5-BODIPY FL localization in mitochondria (green). Mitochondrial marker Mitotracker Red CMSRos (red) was merged, showing the mitochondrial colocalization (yellow). (F) Colocalized area of BODIPY-MA-5 FL or BODIPY itself with MitoTracker Red CMSRos–stained mitochondrial area (1 nM; n=3). *P<0.05 (unpaired two–tailed t test). (G) Fluorescent intensity of MA-5-BODIPY FL in the mitochondrial region after pretreatment with various concentrations (0.3–30 μM) of MA-5 or IAA (n=3). *P<0.05 (unpaired two–tailed t test versus DMSO). (H) Intracellular ATP measurement of Hep3b cells transfected with a mitofilin expression vector or control vector after administration of 0.1% DMSO vehicle or MA-5 (10 μM). *P<0.05; ***P<0.001 (two-way ANOVA and Tukey post hoc test; n=4). RLU, relative luciferase unit; RU, response unit.

We further confirmed the specific binding of MA-5 with mitofilin by liquid chromatography (LC) /MS/MS–based kinetic analysis. MA-5 reacted stoichiometrically with mitofilin, and Scatchard plots revealed Bmax=12.5±4.35 ng and Kd=28.2±11.4 μM (Figure 2C).

For further confirmation, MA-5–mitofilin interaction was evaluated by surface plasmon resonance (SPR) monitoring. Because mitofilin is a membrane protein and because the full-length protein was highly resistant to solubilization by the various detergents tested, we prepared a truncated mitofilin lacking the N–terminal transmembrane domain (mitofilin d1–120) to conjugate it with Biacore sensor chips (Figure 2D). MA-5 dose dependently bound to mitofilin d1–120. The binding affinity was relatively low (Kd=364 μM) compared with LC/MS/MS. This may depend on the construct used and aggregation of the recombinant protein.

To examine the subcellular localization of MA-5, fluorescence–labeled MA-5 (BODIPY-MA-5) was synthesized11 and reacted with normal human fibroblasts (Figure 2E, left panel). Fluorescence signals of BODIPY-MA-5 (green in Figure 2E, right panel) (10 nM) were clearly detected in the cytoplasm and colocalized (yellow in Figure 2E, right panel) with mitochondria labeled by Mitotracker (red in Figure 2E, right panel). By monitoring the pharmacokinetics with a cell–based imaging system, 43% of the BODIPY-MA-5 was localized in the mitochondrial area, whereas only 4% of BODIPY itself was detected (Figure 2F). Under this condition, pretreatment with MA-5 inhibited the accumulation of BODIPY-MA-5 into mitochondria (Figure 2G, left panel). However, IAA had no effect on the BODIPY-MA-5 accumulation (Figure 2G, right panel). Therefore, MA-5 seemed to converge primarily onto mitochondria, suggesting the specific binding to mitochondria.

To further investigate the functional interaction of mitofilin with ATP production, mitofilin cDNA was transfected into Hep3B cells, and the ATP content was measured in the presence or absence of MA-5 (Figure 2H). The overexpression of mitofilin significantly increased the basal ATP level, and the increased ATP level was further elevated by MA-5 in cells that overexpressed mitofilin.

Mutant mice bearing mitochondrial mutations could provide useful models for studies on mitochondrial diseases. Although many lines of mitochondrial disease model mice were generated by disrupting nuclear DNA–coded mitochondrial proteins, recently, we generated mutant mice, namely Mitomice, which carry deleted mitochondrial DNA (ΔmtDNA), resulting in a respiration defect representative of a human disease phenotype.12 However, compared with disease model mice generated by manipulation of the nuclear genome, Mitomice are not suitable for large-scale studies. Each mouse has to be generated by implanting mutated mtDNA into fertilized eggs, because a ΔmtDNA molecule can be inherited only in the first pregnancy, and the mouse cannot be maintained as a strain. Accordingly, we have only a limited number of Mitomice∆ to use for our experiments. We first selected three Mitomice carrying the same loads of mutant mtDNA in the tail DNA (53%–57%) and examined the effect of MA-5 on their lifespan. A control Mitomouse administered DMSO died at around 9 months, whereas the two MA-5–administered Mitomice survived to 11 months (Figure 3A, case 1).

Figure 3.
Figure 3.

MA-5 improved respiration of cardiac and renal cell in Mitomice. (A) Lifespan of Mitomice with the administration of MA-5 at 50 mg/kg body wt per day or vehicle in corn oil (case 1). The percentage of ΔmtDNA4696 in each Mitomouse was determined. (B) Vehicle or MA-5 at 50 mg/kg body wt per day was gavaged for 2 months, and succinate dehydrogenase (SDH) and COX activity were assessed by immunostaining of the heart tissues from Mitomice (cases 2 and 3). Proportion of COX/SDH-positive cells was calculated. **P<0.01 (unpaired two–tailed t test versus vehicle control). (C) Mitochondrial respiration in the kidney tubules. (D) Schematic model of the action of MA-5. MA-5 interacts with mitofilin and modifies the MINOS complex. MA-5 directly binds to mitofilin. It is reported that mitofilin interact with F1FO ATP synthase,23,24 and MA-5 may also directly or indirectly interact with F1FO-ATPase, RuvBL1/RuvBL2, VDAC, HSP70, and CHDCD3 (all putative interacting molecules with MA-5 are colored in red). Modified from Koob and Reichert13 and Darshi et al.14, with permission. ATPase, ATP synthase; CJ, cristae junction; F1F0, F1 subunit and F0 subunit of ATP synthase.

To examine mitochondrial respiration defects, Mitomice carrying the same densities were paired and divided into two groups for oral administration of DMSO or MA-5 (Figure 3B). After treatment for 2 months, the complexes II (succinate dehydrogenase) and IV (cytochrome c oxidase [COX]) were immunochemically examined. In mtDNA-based diseases, cells show respiration defects only when mutant mtDNA was accumulated because of the threshold effects. The mosaic pattern of COX–positive (brown in Figure 3B) and –negative (blue in Figure 3B) cardiac cells can be seen in tissues carrying deleted mtDNA.12 In the case 2 study using a Mitomice pair, cardiac muscle from the DMSO-treated mouse (50%) exhibited a mosaic pattern of blue and brown colors, whereas healthy myocytes were predominant in the paired MA-5–administered mouse, although with high mutation density (69%) (Figure 3B). In the case 3 study using Mitomice bearing the mutation at high mutation densities in the heart (85% and 84%), more than one half of the myocytes were defective in the heart of the control Mitomice, but the MA-5–treated Mitomouse showed mainly healthy cardiomyocytes (Figure 3B). In cases 2 and 3, the proportion of blue cells in the cardiac muscle was significantly decreased by MA-5 treatment (P<0.05) (Figure 3B, lower panel). In the Mitomice kidney, mitochondrial respiration in the nephrons was remarkably increased in MA-5–treated Mitomice compared with the DMSO-treated group with high mutation densities (Figure 3C). These data suggested that MA-5 improved mitochondrial COX activity and respiration in mitochondrial damaged heart and kidney.

In this study, we revealed that MA-5 targets the mitochondrial protein mitofilin, facilitating ATP production and rescuing cardiac and renal mitochondrial respiration.

Mitofilin forms a core complex in the mitochondrial inner membrane organizing system (MINOS), which is composed of mitofilin, MINOS1, CHCHD3, CHCHD6, APOOL, and APOO13 (Figure 3D). The MINOS complex is reported to interact with HSP70 and CHCHD3.14 MINOS is required for keeping crista membranes attached to the inner membranes by crista junctions,15 which form the crista barrier that limits the diffusion of metabolites and modulates the pH gradient across the inner membrane to regulate OXPHOS.16 Deficiencies of mitofilin,17 ChChd3,14 and CHCHD618 cause a decrease in the ATP levels. Thus, it is suggested that MA-5 may modulate ATP synthesis through the MINOS complex.

Our data also revealed that MA-5 does not exert any evident influence on mitochondrial OXPHOS, which is responsible for >90% of ATP production.19 One of the possible mechanisms of MA-5 is an effect on the olgomerization of ATP synthase. The oligomerization of ATP synthase is essential for the maintenance of crista junctions,20 and it increases the local pH gradient or membrane potential, optimizing ATP synthesis without changing the mitochondrial membrane potential.21 The oligomerization depends on two subunits of FO part, subunit e (Su e) and subunit g (Su g). These two subunits are involved in generating the mitochondrial cristae morphology and the assembly of ATP synthase dimer, thereby controlling the mitochondrial biogenesis.22 It was reported that the mitofilin homolog Fcj1 antagonizes the action of Su e and Su g and inhibits the oligomerization of ATP synthase.23,24 Thus, it is postulated that MA-5 may interfere with the interaction between mitofilin and Su e and/or Su g and regulate the dimer formation after the activation of ATP synthesis.

In addition, MA-5 interacts with HSP70, ATP synthase subunits-α and -β, RuvBL1 and -2, VDAC1, and CHCHD3 (Figure 2A). It is possible that the immunoprecipitation of such proteins by MA-5–conjugate FG beads may occur through the interaction of MA-5 with mitofilin. In addition, HSP70 itself is characterized by ATPase activity.25 RUVBL1 and RUVBL2 are ATP-binding proteins.26 VDAC possesses one major binding region for ATP.27 Because indoles have some structural homology to the adenosine group of ATP,28 our data further suggest the possibility that MA-5 may directly interact within these molecules and exert its function. Recently, Uchihashi et al.29 reported that ATP itself caused conformational transitions using purified α3β3-subcomplex sufficient as the motor of ATP hydrolysis. Additional experiments are necessary to reveal these effects of MA-5 on mitofilin and ATP production.

In conclusion, MA-5 may represent a novel therapeutic strategy for treating AKI, CKD, and SRNS as a mitochondria-homing drug.

Concise Methods

Animal Experiments

All animal experiments were approved by the Tohoku University and Harvard Medical School Animal Care Committees. MA-5 was administered at 50 mg/kg body wt by gavage in corn oil 3 hours before ischemia surgery. Ischemia was induced by a retroperitoneal approach to both kidneys of C57/BL 6 male mice for 26 minutes at 37°C as previously reported.7 In mouse CIN, cisplatin was administered at 20 mg/kg body wt by intraperitoneal injection, and MA-5 was administered at 50 mg/kg per day by gavage. Plasma creatinine and BUN were determined by the picric acid method (Beckman Creatinine Analyzer 2 Model7) and Infinity Urea Liquid Stable Regent (Thermo Fisher Scientific), respectively.

Forty-eight hours after ischemia surgery or 96 hours after cisplatin injection, mice were euthanized, and the kidneys were fixed in Methacarn solution for 24 hours. Kidney sections were embedded in paraffin, and 5-μm sections were cut and stained with Periodic acid–Schiff stain for histology examination. Histologic changes caused by tubular necrosis, loss of brush border, cast formation, and dilation were as follows: 0, none; 1, ≤10%; 2, 11%–25%; 3, 26%–45%; 4, 46%–75%; and 5, >76%. This analysis was performed in a blinded fashion, and 10 random visual fields (×200) in the renal outer medullar area were analyzed per kidney section.

Immunohistochemistry staining of the kidney was performed on paraffin sections as previously described.7 Briefly, Methacarn–fixed paraffin sections were deparaffinized, rehydrated, and labeled with goat anti–mouse KIM-1 polyclonal antibody (1:400 dilution; AF1817; R&D Systems) followed by biotinylated donkey anti–goat IgG (1:400 dilution; Jackson ImmunoResearch Laboratories) and ABC peroxidase DAB labeling. ImageJ 1 1.48v soft software was used to measure KIM-1–positive areas, and the analysis was performed in 10 random visual fields (×200) in the renal outer medullar area per kidney section.

For evaluation of the blood concentrations of MA-5, MA-5 was orally administered at doses of 25, 50, or 150 mg/kg to C57/BL 6 mice, and blood samples were collected at the designated times. After 1 hour, the mice were euthanized. The blood concentration of MA-5 was determined by LC/MS/MS on the basis of previously published methods.6 Briefly, the LC/MS/MS system consists of a NANOSPACE SI-II HPLC equipped with a dual-pump system, autosampler, and column oven (Shiseido, Tokyo, Japan) and a TSQ Quantum Ultra (Thermo Fisher Scientific) Triple Quadrupole Mass Spectrometer equipped with a heated electrospray ionization source. LC separation was performed using an Xbridge C18 (100×2 mm inner diameter; 3.5-μm particle size; Waters) with a 40°C oven temperature. The concentrations of the individual compounds were calculated from a regression of the calibration curves.

Mitomice

A transmitochondrial mouse model (Mitomice) carrying wild-type mtDNA and mutant mtDNA with a pathogenic 4696-bp deletion (ΔmtDNA) was generated by Kazuto Nakada at the University of Tsukuba.12 In the lifespan assessment of Mitomice, Mitomice were fed with normal chow and MA-5 at 50 mg/kg body wt per day or vehicle corn oil. For histochemical analysis, Mitomice hearts were excised from the animals, and 10-μm cryosections from the tissues were stained for complexes II (succinate dehydrogenase) and IV (COX) activity as previously reported.12

Measurement of Oxygen Consumption in Mitochondria

Mitochondrial oxygen consumption was measured as previously described.30 Mitochondria were energized with 4 mM succinate to initiate respiration. State 3 oxygen consumption was obtained using 70 μM ADP, and the following state 4 oxygen consumption was measured as the oxygen consumption rate with no ATP synthesis in the absence of ADP.

Measurement of Mitochondrial ROS Production

Mitochondrial ROS (H2O2) production rates were determined fluorometrically by measurement of oxidation of 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red; ex/em=544/590 nm) coupled to the enzymatic reduction of H2O2 by horseradish peroxidase in the presence of exogenous superoxide dismutase. Values are means±SEMs of data from individual mitochondrial samples (P<0.05 versus DMSO treatment).

Affinity Purification Using FG Beads

Magnetic FG beads10 were incubated with 10 mM MA-5 or vehicle DMSO and 1 M potassium carbonate in 1 ml N,N-dimetylformamide for 24 hours at 60°C. Unreacted residues were masked using 20% carbonic anhydride in N,N-dimetylformamide according to the manufacturer’s instruction (Tamagawa Seiki Co., Ltd.). MA-5–immobilized and control beads were equilibrated with binding/wash buffer. Cell extracts were obtained from Hep3B cells according to the Dignam method and incubated with beads. After washing, the bound proteins were eluted and subjected to SDS-PAGE and silver staining.

The SDS-PAGE bands were subjected to in-gel digestion by trypsin after alkylation with acrylamide. The peptide samples were loaded onto a 75-μm fused silica capillary column containing C18 resin. The peptides were eluted with an acetonitrile gradient (typically 2.5%–40%) in 0.1% formic acid and analyzed by an LTQ Orbitrap XL Mass Spectrometer (Thermo Fisher Scientific). A database search on NCBInr and SwissPlot databases was performed using the MASCOT, version 2.4.00 search engine (Matrix Science). Protein identification was considered when the MASCOT score was greater than the criteria (P<0.05).

SPR

SPR was performed in a Biacore T200 Instrument to analyze the interaction between Mitofilin d1–120 and MA-5. Mitofilin d1–120 was immobilized by capture with an anti-His antibody (Qiagen) at a level of about 1200 RU on sensor chip CM5 (GE Healthcare). The binding of MA-5 to Mitofilin d1–120 was obtained by injecting increasing concentrations of MA-5 into the sensor chip under the buffer condition with PBS containing 0.005% (v/v) Tween-20, pH 7.4, at a flow rate of 30 ml min−1 at 25°C. The data were corrected by subtracting the responses from a blank flow cell in which the anti-His antibody was immobilized. The binding responses were plotted in terms of the concentration of MA-5, and the binding affinity (Kd value) was calculated with Biacore T200 Evaluation software (GE Healthcare).

Assay of BODIPY–Based Fluorescent–Conjugated MA-5 Contents in Mitochondria

Human normal fibroblasts were pretreated with MA-5 before the addition of BODIPY–based fluorescent–conjugated MA-5 (MA-5-BODIPY FL). After incubation with 100 nM MA-5-BODIPY FL, the cells were washed with PBS, Hoechst 33342, and MitoTracker Red CMXROS dissolved in PBS for nuclear and mitochondria staining. The cells were imaged and analyzed with a Cellomics ArrayScan VTI HCS Reader (Thermo Fisher Scientific). The fluorescence intensity of MA-5-BODIPY FL in mitochondria was analyzed using the Thermo Scientific Cellomics Colocalization BioApplication.

Statistical Analyses

The data were expressed as means±SEMs. Comparisons were made using unpaired two–tailed t tests, one-way ANOVA, and Dunnett test or two-way ANOVA and Tukey post hoc test as appropriate (JMP Pro 11 software). A P value of <0.05 was considered significant (*P<0.05; **P<0.01; ***P<0.01).

Disclosures

None.

Acknowledgments

We thank Toshiaki Yamamoto, Sadamitsu, Ichijo, Hajime Ikenouchi, Tohihito Sahara, Yusuke Suzuki, Shun Sasaki, Kosuke Suzuki, Kokyo Sakurada, and Yasuno Mukouyama for technical assistance.

This work was supported, in part, by National Grant-in-Aid for Scientific Research 26670070 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and Translational Research Network Program B20.

Footnotes

  • T. Suzuki, H.Y., and M. Kikusato contributed equally to this work.

  • Published online ahead of print. Publication date available at www.jasn.org.

  • See related editorial, “Mitochondria in Kidney Injury: When the Power Plant Fails,” on pages 1869–1872.

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Mitochonic Acid 5 Binds Mitochondria and Ameliorates Renal Tubular and Cardiac Myocyte Damage
Takehiro Suzuki, Hiroaki Yamaguchi, Motoi Kikusato, Osamu Hashizume, Satoru Nagatoishi, Akihiro Matsuo, Takeya Sato, Tai Kudo, Tetsuro Matsuhashi, Kazutaka Murayama, Yuki Ohba, Shun Watanabe, Shin-ichiro Kanno, Daichi Minaki, Daisuke Saigusa, Hiroko Shinbo, Nobuyoshi Mori, Akinori Yuri, Miyuki Yokoro, Eikan Mishima, Hisato Shima, Yasutoshi Akiyama, Yoichi Takeuchi, Koichi Kikuchi, Takafumi Toyohara, Chitose Suzuki, Takaharu Ichimura, Jun-ichi Anzai, Masahiro Kohzuki, Nariyasu Mano, Shigeo Kure, Teruyuki Yanagisawa, Yoshihisa Tomioka, Masaaki Toyomizu, Kohei Tsumoto, Kazuto Nakada, Joseph V. Bonventre, Sadayoshi Ito, Hitoshi Osaka, Ken-ichi Hayashi, Takaaki Abe
JASN Jul 2016, 27 (7) 1925-1932; DOI: 10.1681/ASN.2015060623
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