Abstract
Mitochondrial dysfunction is both a cause and target of reactive oxygen species during ischemia-reperfusion, drug, and toxicant injury. After injury, renal proximal tubular cells (RPTC) recover mitochondrial function by increasing the expression of the master regulator of mitochondrial biogenesis, peroxisome-proliferator-activated-receptor-γ-coactivator-1α (PGC-1α). The goal of this study was to determine whether 5-hydroxytryptamine (5-HT) receptor agonists increase mitochondrial biogenesis and accelerate the recovery of mitochondrial function. Reverse transcription-polymerase chain reaction analysis confirmed the presence of 5-HT2A, 5-HT2B, and 5-HT2C receptor mRNA in RPTC. The 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI; 3–10 μM) increased PGC-1α levels, expression of mitochondrial proteins ATP synthase β and NADH dehydrogenase (ubiquinone) 1β subcomplex 8 (NDUFB8), MitoTracker Red staining intensity, cellular respiration, and ATP levels through a 5-HT receptor and PGC-1α-dependent pathway. Similar effects were observed with the 5-HT2 agonist m-chlorophenylpiperazine and were blocked by the 5-HT2 antagonist 8-[3-(4-fluorophenoxy) propyl]-1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one (AMI-193). In addition, DOI accelerated the recovery of mitochondrial function after oxidant-induced injury in RPTC. This is the first report to demonstrate 5-HT receptor-mediated mitochondrial biogenesis, and we suggest that 5-HT-agonists may be effective in the treatment of mitochondrial and cell injury.
Mitochondrial dysfunction is a common consequence of ischemia-reperfusion, drug, and toxicant-induced renal injury (Campbell and al-Nasser, 1996; Feldkamp et al., 2005; Honda et al., 2005). It is interesting to note that oxidant-induced mitochondrial dysfunction recovers spontaneously in primary cultures of renal proximal tubular cells (RPTC) in culture, demonstrating the presence of an endogenous mechanism to promote the recovery of mitochondrial function (Nowak et al., 1998; Rasbach and Schnellmann, 2007b). For example, after t-butyl-hydroperoxide (TBHP) exposure, RPTC mitochondria undergo lysosomal autophagy, effectively reducing total RPTC mitochondrial volume (Rasbach and Schnellmann, 2007b). The recovery of mitochondrial function in RPTC after oxidant injury is temporally related to expression of the mitochondrial biogenesis regulator peroxisome-proliferator-activated-receptor-γ-coactivator-1α (PGC-1α) (Puigserver et al., 1998; Scarpulla, 2002; Rasbach and Schnellmann, 2007b). The expression of PGC-1α after oxidant injury in RPTC is dependent upon the activation of Src, p38 mitogen-activated protein kinase, and the epidermal growth factor receptor signaling cascades that have also been implicated in postinjury migration and proliferation responses (Yano et al., 1999; Zhuang et al., 2005).
To determine whether increased mitochondrial biogenesis before oxidant injury was protective or potentiated oxidant injury, adenoviral overexpression of PGC-1α in RPTC was used to activate the mitochondrial biogenesis program and increase mitochondrial content before oxidant injury. Depending on the oxidant, increased mitochondria either had no effect or potentiated cellular injury. In contrast, increased mitochondrial biogenesis after oxidant injury accelerated the recovery of mitochondrial functions and ATP-dependent cellular functions (Rasbach and Schnellmann, 2007a). We suggest that targeting mitochondrial biogenesis is a viable therapeutic option to promote the recovery of mitochondrial and cellular functions after injury (Rasbach and Schnellmann, 2007a,b, 2008).
Several signaling molecules have been shown to regulate the expression of PGC-1α in response to a variety of stimuli, including nitric oxide, p38 mitogen-activated protein kinase, the tyrosine kinase Src, the epidermal growth factor receptor, cAMP response element-binding protein, activating transcription factor-2, myocyte enhancer factor-2, calcium/calmodulin-dependent protein kinases, calcineurin A, AMP-activated protein kinase, and SIRT1 (Herzig et al., 2001; Schaeffer et al., 2004; Nemoto et al., 2005; Jäger et al., 2007). Because PGC-1α is responsive to stimuli such as cold exposure, lipopolysaccharide, oxidative stress, caloric restriction, adrenergic stimulation, exercise, and mitochondrial dysfunction, we believed that PGC-1α expression and activity may be amenable to pharmacological manipulation (Herzig et al., 2001; Zong et al., 2002; Suliman et al., 2004; Akimoto et al., 2005; Nemoto et al., 2005; Rasbach and Schnellmann, 2007b).
Several groups have developed strategies designed to increase the expression and activity of PGC-1α. Recent approaches have targeted the protein deacetylase SIRT1, a known PGC-1α activator (Baur et al., 2006; Lagouge et al., 2006; Rasbach and Schnellmann, 2008). Pharmacologic activation of SIRT1 promotes the deacetylation of PGC-1α and leads to an increase in mitochondrial number and function. For example, we reported several differentially substituted isoflavones and isoflavone derivatives promote mitochondrial biogenesis through the activation of SIRT1 and the subsequent deacetylation and activation of PGC-1α. However, such responses only occur with either high doses or longer exposure times (48 h) (Baur et al., 2006; Lagouge et al., 2006; Rasbach and Schnellmann, 2008). Furthermore, many known SIRT1 activators are naturally derived compounds that have very low bioavailability and therefore are not favorable therapeutic candidates (Karakaya, 2004).
Spiegelman and colleagues recently developed a quantitative PCR screen to identify inducers of PGC-1α expression (Arany et al., 2008). This screening technique demonstrated that PGC-1α expression can be regulated by microtubule and protein synthesis inhibitors (Arany et al., 2008). Although these compounds do not harbor significant therapeutic potential, the technique demonstrates the ability to screen for inducers of PGC-1α, and screening larger libraries may ultimately provide new useful inducers of PGC-1α.
Because the role of 5-HT receptors in mitochondrial biogenesis has not been fully investigated and a receptor-targeted approach to increasing mitochondrial biogenesis is an attractive therapeutic strategy, we hypothesized that 5-HT receptor agonists may produce mitochondrial biogenesis in a variety of organs, including the kidney. Harris and colleagues have recently identified and characterized a putative intrarenal serotonergic system and have shown proximal tubules to contain 5-HT receptors, including type 2B, using RT-PCR (Xu et al., 2007).
Materials and Methods
Reagents.
AMI-193 was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Antibodies to ATP synthase-β subunit, NDUFB8, and glyceraldehyde-3-phosphate dehydrogenase were purchased from Abcam (Cambridge, MA), Invitrogen (Carlsbad, CA), and Fitzgerald Antibodies (Concord, MA), respectively. The antibody to PGC-1α (H300) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). We have reported previously the mitochondrial complex 1 protein ND6 as a marker of mitochondrial biogenesis (Rasbach and Schnellmann, 2007a,b, 2008). We have recently been informed by the manufacturer of the antibody (Invitrogen), that it has been confirmed that the former ND6 antibody actually recognizes the mitochondrial complex 1 protein NDUFB8. Thus, NDUFB8 is a marker of mitochondrial biogenesis. All secondary antibodies were purchased from Pierce Chemical (Rockford, IL). Anti-PGC-1α was used at 1:300, whereas all other antibodies were used at 1:1000.
Isolation and Culture of Renal Proximal Tubules.
Female New Zealand White rabbits (2 kg) were purchased from Myrtle's Rabbitry (Thompson Station, TN). RPTC were isolated using the iron oxide perfusion method and grown in 35-mm tissue culture dishes under improved conditions as described previously (Nowak and Schnellmann, 1996). The culture medium was a 1:1 mixture of Dulbecco's modified Eagle's medium/Ham's F-12 (without glucose, phenol red, or sodium pyruvate) supplemented with 15 mM HEPES buffer, 2.5 mM l-glutamine, 1 μM pyridoxine HCl, 15 mM sodium bicarbonate, and 6 mM lactate. Hydrocortisone (50 nM), selenium (5 ng/ml), human transferrin (5 μg/ml), bovine insulin (10 nM), and l-ascorbic acid-2-phosphate (50 μM) were added daily to fresh culture medium. Confluent RPTC were used for all experiments. RPTC monolayers were treated with various compounds or diluent (dimethyl sulfoxide) for 24 h.
Reverse Transcription-Polymerase Chain Reaction.
Total RNA extraction from RPTC was performed using TRIzol RNA extract reagent (Invitrogen). RNA (3 μg) was subjected to first-strand cDNA synthesis using oligo(dT)18 and ThermoScript Reverse transcriptase (Invitrogen) in a 20-μl total reaction volume. Amplification of cDNA (2 μl) was carried out using GoTaq Green Master mix (Promega, Madison, WI) with 5-HT2 receptor subtype-specific sense and antisense primers (Table 1). Thermal cycling conditions were started with one cycle at 94°C for 30 and then 57°C for 30 s for primer annealing, 72°C for 45 s, and final extension for 10 min at 72°C. PCR was performed using a programmed thermocycler (Eppendorf AG, Hamburg, Germany). The number of amplification cycles and their fragment size are presented in Table 1. The amplified PCR products were subjected to electrophoresis using a 1.8% agarose gel and stained with ethidium bromide.
NRK-52E Cell Experiments.
Normal rat kidney (NRK-52E) epithelial cells were cultured as described previously (Covington et al., 2005) in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal calf serum. For measurement of mitochondrial biogenesis, NRK52-E cells were treated with 10 μM DOI or vehicle for 24 h, stained with MitoTracker Red (200 nM) (Invitrogen) for 20 min, washed, and imaged by fluorescence microscopy. MitoTracker staining intensity was quantified using stand-alone imaging software (Alpha Innotech, San Leandro, CA). To measure PGC-1α transcription, NRK-52E cells were transfected with the 2-kb PGC-1α promoter with a firefly luciferase reporter (a generous gift from Bruce Spiegelman, Harvard Medical School, Boston, MA) and the constitutive pGL4.7[hRluc/TK] Renilla reniformis reporter as a control for transfection efficiency. DOI was added 48 h after infection to allow for sufficient expression of reporter vectors. Promoter activity was then measured 24 h later using a dual-luciferase reporter assay system (Promega).
Oxygen Consumption.
RPTC bathed in 37°C culture medium were gently detached from culture dishes with a rubber policeman and transferred to a 37°C QO2 chamber 48 h after the initial exposure to the compounds. Basal and uncoupled (FCCP; 1 μM) RPTC QO2 was measured polarographically using a Clark-type electrode as described previously (Nowak and Schnellmann, 1996).
Adenoviral PGC-1α RNAi.
PGC-1α RNAi plasmid was a generous gift from Marc Montminy (Salk Institute for Biological Studies, San Diego, CA) (Koo et al., 2004). The plasmid was digested by the endonuclease restriction enzyme PAC I (New England Biolabs, Ipswich, MA) for 5 h to linearize the Ad-Track PGC-1 RNAi plasmid. Linearized plasmid was transfected into human embryonic kidney 293 cells, and the virus was propagated for 7 days and scaled up until sufficient quantities for infection were obtained. Virus was titrated to achieve 100% green fluorescent protein-positive RPTC. Virus was present in media for 24 h. Knockdown of PGC-1α was measured 48 h after initial exposure to PGC-1 RNAi in the presence of the proteasome inhibitor MG-132 for 4 h to stabilize PGC-1α expression.
Cell Number.
Measurement of monolayer protein content over time was used to estimate cell number. RPTC monolayers were washed with PBS, solubilized in Triton buffer (0.05% Triton X-100, 100 mM Tris-base, and 150 mM NaCl, pH 7.5), and sonicated for 60 s, and then protein concentrations were determined by the bicinchoninic acid method according to the manufacturer's instructions (Pierce Chemical).
Preparation of Cell Lysates and Immunoblot Analysis.
RPTC were washed twice with PBS without Ca2+ and Mg2+ and harvested in cell lysis buffer from BioVision (Mountain View, CA). Before immunoblot analysis, all cells were disrupted by sonication for 30 s. Samples were boiled and prepared for electrophoresis. Equal amounts of cellular protein lysates were separated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes. After treatment with 5% skim milk or bovine serum albumin at 4°C overnight, membranes were incubated with various antibodies for 2 h and then incubated with an appropriate horseradish peroxidase-conjugated secondary antibody for 1 h. Bound antibodies were visualized by chemiluminescence detection on an imaging system (Alpha Innotech).
Measurement of ATP.
ATP was measured via luciferase-mediated bioluminescence as described previously (Lundin et al., 1986). In brief, RPTC were washed three times with ice-cold PBS and subsequently lysed on ice with Triton/glycylglycine lysis buffer. Homogenates were immediately centrifuged at 14,000g for 5 min at 4°C. Supernatants were collected and kept on ice. Each sample was combined with an equal amount of luciferase reagent (ATP Bioluminescence Assay kit CLS II; Roche Diagnostics, Indianapolis, IN) and imaged immediately using an imaging system (Alpha Innotech).
Statistical Analysis.
Data are presented as means ± S.E. and were subjected to one- or two-way analysis of variance as appropriate. Multiple means were compared using Student-Newman-Keuls test, with p < 0.05 considered to be a statistically significant difference among means. Renal proximal tubules isolated from an individual rabbit represents a single experiment (n = 1). Experiments were performed with two to five plates of cells and repeated until a number of at least three was reached.
Results
DOI (Fig. 1a) is a selective agonist of 5-HT2 (Dedeoğlu and Fisher, 1991; Cumming-Hood et al., 1993). Using RT-PCR, we confirmed that primary cultures of RPTC express the 2A, 2B, and 2C subtypes of the 5-HT receptor (Fig. 1b).
Increasing concentrations of DOI increased activity of the 2-kb PGC-1α promoter, suggesting 5-HT receptors may mediate the transcription of this mitochondrial biogenesis regulator (Fig. 2a). To determine the effects of activating 5-HT2 receptors on mitochondrial biogenesis, increasing concentrations of DOI were added to RPTC for 24 h, and expression of mitochondrial proteins, ATP synthase β, and NDUFB8 were measured as markers of mitochondrial content (Fig. 2b). Increases in mitochondrial protein expression were seen at 3 μM DOI and were maximal at 10 μM DOI, suggesting that activation of 5-HT receptors with DOI induces mitochondrial biogenesis.
To further verify DOI-induced mitochondrial biogenesis, a rat epithelial cell line (NRK-52E) was used. NRK-52E cells were exposed to DOI (10 μM) for 24 h and then stained with the mitochondria-specific dye MitoTracker Red (Fig. 3a). Fluorescence microscopy revealed an ∼2-fold increase in MitoTracker staining, again suggesting that DOI is capable of inducing the mitochondrial biogenesis program in renal cells (Fig. 3b).
RPTC treatment with DOI promoted a concentration-dependent increase in both basal and uncoupled respiration and cellular ATP concentrations (Fig. 4). Lower concentrations of DOI (1 and 3 μM) did not effect basal respiration; however, the higher concentration (10 μM) increased basal respiration by ∼30% (Fig. 4a). Both 1 and 3 μM DOI increased uncoupled respiration, with maximal increases at 10 μM (Fig. 4b). Similar to the changes in basal respiration, ATP concentrations were not affected at DOI concentrations of either 1 or 3 μM, but they were increased ∼45% by 10 μM DOI (Fig. 4c). These data reveal that DOI promotes the biogenesis of functional mitochondria in RPTC. Consistent with these findings, RPTC exposed to 10 μM m-chlorophenylpiperazine, another 5-HT2 receptor agonist, resulted in elevated FCCP-uncoupled respiration (130 ± 5% control) at 24 h (data not shown).
Because the mitochondrial biogenesis program is known to be initiated in response to a number of cell stresses (Suliman et al., 2004; St-Pierre et al., 2006; Rasbach and Schnellmann, 2007b; Spiegelman, 2007), total monolayer protein content was measured to ensure that DOI did not initiate proliferation or exhibit toxicity. None of the concentrations of DOI had any effect on monolayer protein content, suggesting that DOI was not overtly toxic nor induced RPTC proliferation under these experimental conditions (Fig. 4d).
To determine whether DOI increased PGC-1α and to verify that DOI acts through a 5-HT receptor, RPTC were pretreated with the pan-5-HT receptor antagonist AMI-193 (10 μM), and samples were analyzed for PGC-1α expression 12 h post-DOI (10 μM) exposure (Fig. 5). PGC-1α expression was up-regulated in DOI-treated RPTC; however, the DOI-mediated increase in PGC-1α expression was attenuated by AMI-193, suggesting that DOI induced increases in PGC-1α expression are mediated by 5-HT receptors.
To validate that DOI-induced mitochondrial biogenesis was dependent on PGC-1α, PGC-1α expression was first decreased ∼45% 48 h after infection with an adenoviral PGC-1α-RNAi compared with a scramble RNAi control (Fig. 6a). RPTC were then exposed to DOI (10 μM) for 24 h in the presence or absence of PGC-1α-RNAi. Exposure to DOI increased the expression of the mitochondrial proteins ATP synthase β and NDUFB8, and this increase in mitochondrial proteins was blocked in RPTC in which PGC-1α had been decreased using RNAi (Fig. 6b). We suggest that DOI-mediated mitochondrial biogenesis is dependent on PGC-1α.
Because increasing PGC-1α expression and mitochondrial biogenesis subsequent to oxidant injury promotes the recovery of mitochondrial and cellular functions (Rasbach and Schnellmann, 2007a), we sought to examine the effects of DOI on RPTC mitochondrial function after exposure to the model oxidant TBHP (200 μM for 30 min). DOI (10 μM) was added to RPTC 6 h after TBHP exposure to allow for the injury process to take place, and respiration was measured 24 h later. TBHP induced mitochondrial dysfunction as reflected by decreases in both basal and uncoupled respiration (Fig. 7). Treatment of RPTC with DOI promoted the return of both basal and uncoupled respiration to control values within 24 h after the initial exposure to TBHP, whereas untreated RPTC still exhibited significant mitochondrial dysfunction (Fig. 7). In contrast, when RPTC were pretreated with DOI (10 μM) for 24 h before TBHP exposure, basal and uncoupled respiration remained decreased 24 h after TBHP exposure, suggesting DOI pretreatment offers no protection from oxidant injury (Fig. 8). These data reveal that DOI-induced increases in mitochondrial biogenesis accelerate the recovery of mitochondrial function after oxidant injury, but they do not confer protection from oxidant-mediated mitochondrial dysfunction.
Discussion
Although mitochondrial dysfunction is a common consequence of ischemic- and drug-induced injuries, and other disease processes, very few therapies have been directed toward preventing or promoting the recovery of mitochondrial injury. Furthermore, mitochondrial number is decreased after oxidative stress observed in models of ischemia-reperfusion injury or from the cumulative oxidative stress associated with aging (Lemasters, 2005; Rasbach and Schnellmann, 2007b). Several disease models or toxicants decrease the expression of PGC-1α and are associated with impaired mitochondrial biogenesis (Portilla et al., 2002; Priault et al., 2005). Therefore, disease states or injury models associated with mitochondrial dysfunction and/or impaired biogenesis of mitochondria is therefore attractive targets for therapeutics aimed at stimulating the mitochondrial biogenesis pathway.
In this study, we describe a novel 5-HT-dependent mechanism controlling the biogenesis of mitochondria. The 5-HT2 receptor agonist DOI increased PGC-1α promoter activity, suggesting that agonism of 5-HT receptors regulates the activity of PGC-1α at the transcriptional level. The increase in PGC-1α promoter activity was observed with 10 μM DOI, and this concentration of DOI maximally increased mitochondrial biogenesis as demonstrated by the concentration-dependent increase in mitochondrial protein expression (ATP synthase β and NDUFB8), MitoTracker Red staining intensity, cellular respiration, and ATP concentrations. At 3 μM, DOI increased some markers of mitochondrial biogenesis. The 5-HT2 receptor agonist m-chlorophenylpiperazine also increased mitochondrial biogenesis. The pan-5-HT receptor antagonist (AMI-193) blocked DOI-mediated increases in PGC-1α, demonstrating that DOI acted through 5-HT receptors. Although DOI is selective for 5-HT2 receptors, the EC50 value for DOI in isolated receptor systems is in the nanomolar range. This observation provides evidence that DOI may act as a more promiscuous activator of 5-HT receptor subtypes in this system and that the observed increases in mitochondrial biogenesis may be through other 5-HT receptors.
Nebigil et al. focused on the role of 5-HT2B receptor signaling in the heart (Nebigil et al., 2003a,b; Nebigil and Maroteaux, 2003). Overexpression of 5-HT2B receptors in the heart lead to increases in mitochondrial enzyme activities in isolated ventricular slices (Nebigil et al., 2003b). In addition, these mice developed severe cardiac hypertrophy and a dilated cardiomyopathy as a result of mitochondrial proliferation and increased cell number and size, which were similar to the phenotype observed in transgenic mice with cardiac specific overexpression of PGC-1α (Lehman et al., 2000; Lehman and Kelly, 2002). Specific knockout of 5-HT2B receptors in the heart resulted in marked decreases in succinate dehydrogenase and cytochrome oxidase activities in ventricular sections and reductions in mitochondrial number associated with increased myocardial cell death (Nebigil et al., 2003a).
Because PGC-1α is known to promote mitochondrial biogenesis in RPTC (Rasbach and Schnellmann, 2007a,b, 2008), we validated that the observed increase in PGC-1α expression was responsible for mediating DOI-induced mitochondrial biogenesis. PGC-1α expression was reduced ∼45% using an adenoviral PGC-1α-RNAi for 48 h. The knockdown of PGC-1α was sufficient to prevent DOI-induced increases in the expression of both nuclear- (ATP synthase β) and mitochondrial (NDUFB8)-encoded mitochondrial proteins, revealing that DOI increases mitochondrial biogenesis through a PGC-1α-dependent mechanism.
Cell stress is often associated with compensatory responses such as mitochondrial biogenesis (Rasbach and Schnellmann, 2007b; Spiegelman, 2007). We determined whether DOI was toxic to RPTC at any concentrations shown to promote mitochondrial biogenesis. Neither morphological changes (data not shown) nor a change in cell number was observed, suggesting DOI did not induce injury in RPTC.
Increasing the expression of PGC-1α after oxidant injury is known to accelerate the recovery of RPTC mitochondrial and cellular functions (Rasbach and Schnellmann, 2007a). Thus, we determined the effect of DOI-mediated increases in PGC-1α expression and mitochondrial biogenesis on the recovery of mitochondrial function after injury. The addition of DOI 6 h post-TBHP exposure increased the rate of recovery of both basal and uncoupled cellular respiration 24 h after injury. Pretreatment of RPTC with DOI for 24 h, however, did not protect against TBHP-induced mitochondrial dysfunction, which is consistent with our previous study that showed overexpressing PGC-1α before oxidant injury did not protect mitochondrial function (Rasbach and Schnellmann, 2007a). Therefore, we suggest that DOI or other 5-HT receptor agonists may be an effective therapeutic option to accelerate the recovery of renal function subsequent to acute kidney injury.
Identifying a receptor-mediated approach to increase the expression of PGC-1α and promote mitochondrial biogenesis may be a viable therapeutic option for the treatment of a variety of disorders associated with mitochondrial dysfunction. Although chronic overexpression of 5-HT2B or PGC-1α in the heart has pathological effects (Lehman et al., 2000; Lehman and Kelly, 2002), limited exposure to pharmacological activators of mitochondrial biogenesis during periods of mitochondrial dysfunction may result in improved tissue function with limited untoward effects. Furthermore, agents that increase 5-HT receptor activation indirectly, such as 5-HT reuptake inhibitors, may act through mitochondrial biogenesis. Finally, targeting specific 5-HT receptors to induce mitochondrial biogenesis may provide some order of specificity for targeting the biogenesis response, as the actions would be limited to those tissues expressing specific 5-HT receptors.
Footnotes
This work was supported by the National Institutes of Health National Center for Research Resources [Grant C06-RR015455] (to the Medical University of South Carolina Animal Facilities); by the National Institutes of Health National Heart, Lung, and Blood Institute [Grant T32-HL007260] (to K.A.R.; and by the National Institutes of Health National Institute of General Medical Sciences [Grant R01-GM084147] (to R.G.S.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.159947
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ABBREVIATIONS:
- RPTC
- renal proximal tubular cell(s)
- TBHP
- t-butyl-hydroperoxide
- PGC-1α
- peroxisome-proliferator-activated-receptor-γ-coactivator-1α
- SIRT1
- silent mating type information regulation 2 homolog Saccharomyces cerevisiae
- PCR
- polymerase chain reaction
- 5-HT
- 5-hydroxytryptamine
- RT-PCR
- reverse transcription-polymerase chain reaction
- AMI-193
- 8-[3-(4-fluorophenoxy) propyl]-1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one
- NDUFB8
- NADH dehydrogenase (ubiquinone) 1β subcomplex 8
- DOI
- 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride
- FCCP
- carbonyl cyanide p-trifluoromethoxyphenylhydrazone
- RNAi
- RNA interference
- MG-132
- N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal
- PBS
- phosphate-buffered saline.
- Received August 5, 2009.
- Accepted October 28, 2009.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics