Renal ischemia-reperfusion (I/R) is a technically unavoidable obstacle to kidney transplantation accounting for a large percentage of delayed graft function and the associated long-term worst prognosis (1–3). Understanding the pathophysiology of renal I/R is essential for developing disease-specific treatments. However, despite the complexity of the disease, the primary energy deficit during the ischemic phase is obvious, and its length dictates the outcome of reperfusion (4). Less obvious, however, is that the energy deficit persists despite reperfusion (4–6). A major reason for this is the fact that reperfusion is characterized by the generation of reactive oxygen species with a resultant destruction of mitochondria (5, 7). Therefore, a strategy targeted at replacing the damaged mitochondria would restore energy metabolism, replete intracellular adenosine triphosphate (ATP) levels, and thereby attenuate I/R-induced renal injury. Stemming from this notion, we investigated an energy-sensing enzyme that has been shown to modulate mitochondrial biogenesis: Sirtuin 1 (Sirt1), as a promising target in the treatment of renal I/R injury.
Sirtuin 1 is a member of seven evolutionarily conserved mammalian homologues of silencing information regulator 2, which was first found to prolong the life of lower level organisms, such as S. cerevisiae, C. elegans and D. melanogaster in response to caloric restriction (8–10). Sirtuin 1 also plays a similar role in caloric restriction and longevity in mice (11). It is ubiquitously expressed throughout most mammalian organs (12). The dependence of Sirt1 on NAD+ for enzymatic deacetylation is what confers its energy-sensing feature (13), thereby linking cellular energy status to various biologic processes, including inflammation (14, 15), apoptosis (16, 17), and energy metabolism (18–20). The role of Sirt1 in the kidneys has been gaining attention (21–23), most recently implicated as the reason for the protective advantage of younger mice over their older counterparts to tolerate renal I/R (17).
In this study, we hypothesized that stimulation of the Sirt1-peroxisome proliferator–activated receptor gamma coactivator 1-alpha (PGC1α) pathway would enhance mitochondrial biogenesis, thereby diminishing the energy deficit after I/R and ultimately attenuating I/R-induced renal injury. To test this hypothesis, we used a previously established rodent model of bilateral renal I/R injury (24). Animals subjected to renal I/R were posttreated with SRT1720, a small molecule allosteric stimulator of Sirt1 (25). The effects of Sirt1 stimulation on I/R-induced renal injury were determined by analyzing various metabolic, oxidative stress, and inflammatory parameters.
Sirt1 Stimulation Attenuates Renal Injury After Renal I/R
Histologic evaluation of the I/R vehicle group revealed marked tubular injury as compared to the control group, exemplified by extensive tubular necrosis and casts, and few dilated tubules lined by flattened tubular epithelium (Fig. 1A). Impressively, SRT1720 reduced the extent and severity of tubular injury (Fig. 1A). The extent of injury, and subsequent protection by SRT1720, after renal I/R is semiquantitatively represented in Figure 1(C). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis detection showed an average of 1.5±0.2, 32.5±2.4, and 12.0 ± 2.5 TUNEL-positive nuclei per high power field (HPF) in the control, vehicle-treated, and SRT1720-treated groups, respectively (P<0.05, all comparisons) as shown in Figure 1(B).
Serum levels of aspartate aminotransferase (AST) and creatinine were used as indicators of renal injury after I/R (26). The levels of these markers were significantly increased in the vehicle group in comparison to the control group (Fig. 1D and E), indicating a severe degree of renal dysfunction after I/R in our model. SRT1720 treatment showed a 38% and 56% decrease in serum levels of AST and creatinine, respectively, in comparison to the vehicle group (P<0.05; Fig. 1D and E).
Sirt1 Stimulation Enhances Energy Metabolism After Renal I/R
To evaluate the energy deficit corollary of renal ischemia, we determined renal tissue ATP levels. At 24 hr of reperfusion, the vehicle group showed a 48% decrease in ATP levels as compared to control, whereas SRT1720 restored ATP levels up to 77% of control (P<0.05; Fig. 2A). We then investigated whether the difference in ATP levels correlated with the preservation of mitochondrial mass which was measured by quantifying mitochondrial DNA copy numbers (27, 28) and the mitochondrial protein succinate dehydrogenase. Indeed, the increase of ATP levels in the treatment group was also accompanied by a preservation of mitochondrial DNA copy numbers when compared to the vehicle group (P<0.05; Fig. 2B). In addition, there was a 53.1% decrease in protein levels of succinate dehydrogenase after I/R, which was increased by 42.2% in the treatment group in comparison to the vehicle group (P<0.05; Fig. 2C). To further confirm the in vivo findings, human kidney epithelial HK-2 cells were shown to increase ATP levels by 49.2%, adjusted by protein concentration (Fig. 2D), and mitochondrial mass per cell by 41.4%, as determined by mean fluorescent intensity using a mitochondrial stain (Fig. 2E and F), in response to 24 hr of SRT1720 treatment. SRT1720 did not affect HK-2 cell proliferation and size as determined by cell count and forward and side scatter in flow cytometric analysis, respectively.
There was a 60% increase in expression of PGC1α, the master regulator of mitochondrial biogenesis (29), in the kidneys from the treatment group, compared to the vehicle group (P<0.05; Fig. 3A). We also proceeded to examine the levels of the downstream effectors of mitochondrial biogenesis: mitochondrial transcription factor A (mtTFA), nuclear respiratory factor 1 (Nrf-1), and Sirtuin 3 (Sirt3), and they were all consistently upregulated by SRT1720 in comparison to the vehicle group (Fig. 3B, C, and D).
Sirt1 Stimulation Reduces Reactive Oxygen Species After Renal I/R
Ischemia-reperfusion generates free radicals, which in turn induces lipid peroxidation; a process whereby free radicals react with polyunsaturated fatty acids causing cellular membrane damage. Malondialdehyde (MDA) formation serves as an indicator of lipid peroxidation in tissues (30, 31). There was a 72% increase in renal MDA levels in the vehicle group in comparison to the control group, whereas its levels were significantly attenuated by SRT1720 after renal I/R (Fig. 4A). SRT1720 treatment also decreased the protein levels of nitrotyrosine by 30% compared to the vehicle group (P<0.05; Fig. 4B). Moreover, SRT1720 significantly decreased both the messenger RNA (mRNA) expression and protein levels of inducible nitric oxide synthase (iNOS) (P<0.05; Fig. 4C and D).
Sirt1 Stimulation Attenuates the Inflammatory Response After Renal I/R
Inflammation is a major source of injury in renal I/R. We therefore examined the effect of SRT1720 on the extent of macrophage infiltration in the kidneys after I/R. Immunofluorescence of F4/80 macrophage staining showed a dramatic decrease in macrophage infiltration in the kidneys 24 hr after renal I/R as compared to the vehicle group (Fig. 5A). This was also accompanied by a 3.5-fold increase in tumor necrosis factor (TNF)-α expression in the vehicle group when compared to control, which was significantly decreased by 1.6-fold in the treatment group as compared to the vehicle group (P<0.05; Fig. 5B). The decrease in macrophage infiltration and TNF-α expression was associated with a decrease in NF-κB activation as evidenced by a 33% decrease in IκB-α degradation in the vehicle group, compared to the control group, which was inhibited by SRT1720 treatment (Fig. 5C). Consistently, there was a 1.8-fold increase in NF-κB phosphorylation in the vehicle group as compared to the control group which was decreased by 25.5% in the treatment group (P<0.05; Fig. 5D).
Renal I/R remains the most common cause of acute kidney injury in the intensive care unit, especially among postsurgical patients and is a major cause of delayed graft function after renal transplantation. Yet the treatment remains purely supportive. The pathophysiology of renal I/R can be summarized by a primary acute energy deficit caused by ischemia, followed by a secondary oxidative and inflammatory stage that ultimately results in cellular death (apoptosis/necrosis) (3, 4). Interestingly, the energy deficit persists even after reperfusion caused by mitochondrial dysfunction (4–6) and loss of mitochondrial mass (7). In the present study, we showed that pharmacologic Sirt1 stimulation exerted a protective effect on the kidneys by means of the enhancement of mitochondrial mass, thereby diminishing the energy deficit after I/R. Our findings are consistent with an earlier study by Saba et al. which demonstrated that targeting the mitochondrial ATP synthase restores ATP levels and attenuates injury after renal I/R (6). Additional evidence by Rasbach and Schnellmann, working on renal proximal tubule cells in vitro, confirmed that enhanced mitochondrial biogenesis by means of the overexpression of PGC1α was protective after oxidative stress (7). Similarly, we also observe that the increase in mitochondrial mass correlates with the increased expression of PGC1α and its downstream mediators after Sirt1 stimulation in rat kidneys after I/R.
The evidence for the protective effects of Sirt1 in the kidneys is accumulating. We demonstrate a significant reduction of apoptosis in the rat kidneys with SRT1720 treatment, suggesting that activation of energy metabolism by SRT1720 might protect renal cells from apoptosis thereby increasing the number of live cells after I/R. Consistent with our TUNEL results, Fan et al. recently showed that Sirt1 inhibited renal cell apoptosis after I/R through its interaction with p53 (17). In addition, we also demonstrate that SRT1720 can increase the ATP levels and mitochondrial mass of uninjured HK-2 cells, suggesting that energy generation and mitochondrial biogenesis of renal cells surviving I/R injury might be further stimulated by SRT1720, thereby likely contributing to the increased ATP levels and mitochondrial mass observed in the SRT1720-treated rats after renal I/R. The detailed mechanisms at the cellular and molecular levels will require further investigation. However, these findings underscore Sirt1 as a highly promising therapeutic target in renal I/R. More importantly, our work promotes the modulation of energy metabolism as a treatment strategy in renal I/R. Further, we demonstrate that Sirt1 stimulation showed beneficial effects when administered after reperfusion, therefore making it more clinically applicable.
SRT1720 is a small molecule activator of Sirt1, which allosterically increases its affinity to acetylated protein substrates (25). In agreement with our results, pharmacologic stimulation of Sirt1 has been shown to enhance mitochondrial biogenesis and rescue renal proximal cells from oxidative injury by means of the increased transcription and deacetylation of PGC1α (27, 32). Active PGC1α also promotes its own transcription (27, 32). Therefore, the increased expression of PGC1α shown here may be explained by a positive transcriptional feedback loop initiated by Sirt1, as previously noted in other models (28, 33). Deacetylation of PGC1α by Sirt1 also activates the transcription of the mitochondrial biogenesis proteins Nrf-1 and mtTFA (18, 29, 33, 34). Nrf-1 regulates the transcription of multiple nuclear gene products with mitochondrial functions, specifically proteins that are related to respiration and ATP synthesis (35). Additionally, Nrf-1 is directly involved in mitochondrial DNA replication (36) and acts as one of the major determinants of mtTFA expression (37), which was also increased by SRT1720 after I/R. mtTFA is a High mobility Group–Box transcription factor that is responsible for the bidirectional transcription of mitochondrial DNA (38–40). Consistent with our results, it has been recently shown that Sirt3 is another downstream effector of PGC1α and is essential for its effect on mitochondrial biogenesis and oxidative stress (41). The manner in which Sirt3 interacts with the aforementioned transcription factors remains to be determined.
The contribution of oxidative stress to I/R-induced organ injury cannot be overlooked (4, 42). Renoprotective effects of Sirt1 in oxidative stress have also been described (21). Ischemia-reperfusion is associated with the generation of free oxygen radicals and nitric oxide, which form the cytotoxic metabolite peroxynitrite. Consequently, the contribution of iNOS as a catalyst of nitric oxide production in renal I/R-induced oxidative injury is well established (42–45). We report here that SRT1720 significantly decreased the mRNA and protein levels of iNOS after renal I/R. Additionally, peroxynitrite has been specifically shown to cause DNA damage and lipid peroxidation (42). Similar processes have been implicated in mitochondrial dysfunction and resultant energy depletion in renal I/R (46). In the present study, treatment with SRT1720 decreased the levels of nitrotyrosine, a marker of peroxynitrite generation (42, 47), and tissue levels of MDA, a marker of lipid peroxidation (30, 31). These findings beg the question as to whether a direct relationship between Sirt1 and iNOS expression exists. An indirect relationship, however, may be inferred; inflammation has been shown to induce iNOS expression specifically through the NF-κB pathway (48).
Inflammation is an integral part of reperfusion injury, characterized by a complex interplay between the adaptive and innate immune response (49). Herein, we demonstrate that pharmacologic Sirt1 stimulation decreased renal macrophage infiltration and tissue levels of TNF-α expression in the kidneys after I/R. This correlated with the inhibition of NF-κB activation shown by the decrease in the degradation of IκB-α and the phosphorylation of NF-κB in the treatment group compared to the vehicle group. The precise mechanism behind this observation requires further investigation. Nevertheless, previous studies have shown that Sirt1 exerts an anti-inflammatory effect by deacetylating NF-κB, thereby preventing its translocation into the nucleus (14, 15). Our findings suggest possible additional mechanisms exist by which Sirt1 can regulate NF-κB in I/R.
The energy-sensing nature of Sirt1 puts it at the heart of the crisis of I/R: energy deficit. In support of this, we show that pharmacologic Sirt1 stimulation restores cellular energy and mitochondrial mass. Arguably, Sirt1 may exert its effect on restoring mitochondrial mass, in part, through a decrease in mitochondrial injury by attenuating inflammation and oxidative stress. Despite this, we have also demonstrated the restoration of mitochondrial mass through the upregulation of mitochondrial biogenesis as a likely contributor to the renoprotective effects of Sirt1 in I/R.
MATERIALS AND METHODS
A Rodent Model of Renal I/R Injury
Male Sprague-Dawley rats (300–350 g; Charles River Laboratories, Wilmington, MA) were randomly assigned into different groups (n=5/group). Rats were anesthetized with isoflurane (Butler Schein, Dublin, OH) inhalation. Using a midline abdominal incision, renal I/R injury was induced by bilateral renal pedicle clamping for 60 min. Then the clamp was removed, and reflow (reperfusion) was visually verified. Venous cannulation was obtained before reperfusion. Rats were intravenously injected with 1 mL of 5 mg/kg BW SRT1720, N-[2-[3-(piperazin-1-ylmethyl)imidazo[2,1-b]:[1,3] thiazol-6-yl]phenyl]quinoxaline-2-carboxamide (ADOOQ Bioscience, Irvine, CA) or 20% dimethyl sulfoxide in normal saline (vehicle) at the start of reperfusion. Control animals were euthanized for sample collection and were not subjected to any surgical procedures (control). At 24 hr after reperfusion, animals were anesthetized, and plasma and tissue samples were harvested and stored at −80°C until analysis. All experiments were performed in accordance with the guidelines for the use of experimental animals by the National Institutes of Health (Bethesda, MD) and were approved by the Institutional Animal Care and Use Committee of the Feinstein Institute for Medical Research.
Determination of Organ Injury Variables
Plasma levels of AST and creatinine were determined at 24 hr after reperfusion by using commercial assay kits according to the manufacturer’s instructions (Pointe Scientific, Lincoln Park, MI).
Morphologic alterations in the kidneys at 24 hr after reperfusion were examined by light microscopy. The tissue was fixed in 10% formalin and later embedded in paraffin. The tissue paraffin sections (4-µm thick) were stained with hematoxylin-eosin. Histological changes in the corticomedullary junction were assessed and scored by a blinded investigator according to a modified score by Kelly et al. (50). Briefly, 10 random HPFs along the corticomedullary junction were examined. Each HPF was evaluated for tubular cell injury (apoptosis, karyorrhexis, and karyolysis), tubular cell detachment, loss of brush border, tubular simplification, and cast formation. Each of the aforementioned categories were scored using a semiquantitative scale: (1) damage involving less than 10% of tubules, (2) area of damage 10% to 25% of tubules, (3) damage involving 25% to 50% of tubules, (4) damage involving 50% to 75% of tubules, and (5) greater than 75% of the kidney affected. An average score for each sample was then calculated.
For TUNEL assay, paraffin-embedded sections were deparaffinized in xylene and rehydrated in a graded series of ethanol. Fluorescence staining was performed using a commercially available In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN). The assay was conducted according to the manufacturer’s instructions. The nucleus was stained with 4′,6-diamidino-2-phenylindole. Results were expressed as the average number of TUNEL-positive staining nuclei per 10 HPFs. For F4/80 macrophage staining, slides were incubated in 0.92% citric acid buffer (Vector Laboratories, Burlingame, CA) at 95°C for 15 min. After cooling for 20 min at room temperature, the slides were incubated with 2% H2O2 in 60% methanol and blocked in 5% bovine serum albumin (BSA)/Tris-buffered saline (TBS), after which they were permeabilized with 0.1% Triton/TBS and incubated with goat anti-F4/80 antibody (1:20; Santa Cruz Biotechnologies, Santa Cruz, CA) in 2.5% BSA/TBS with 0.02% Triton X-100 at 4°C for 1 hr. The slides were washed and incubated with fluorescein isothiocyanate-labeled donkey anti-goat secondary antibody (1:500; Santa Cruz Biotechnologies) in 2.5% BSA/TBS for 1 hr. The slides were mounted with propidium iodide containing mounting medium from Vector Laboratories (Burlingame, CA) and examined under a fluorescent microscope.
Determination of ATP and MDA Levels
Twenty-four hours after reperfusion, the right and left kidneys were divided into sagittal sections. Each half of the kidney was immediately frozen in liquid nitrogen. Later, the frozen right and left kidneys were pulverized together in liquid nitrogen. Kidney tissue (25 mg) was homogenized in 100 µL assay buffer and centrifuged to remove insoluble materials at 13,000g for 10 min. The supernatant was deproteinized by perchloric acid precipitation followed by potassium hydroxide neutralization before subjecting to ATP assay kits from BioVision (Mountain View, CA). For measuring MDA levels, 25 mg of kidney tissue was homogenized in lysis buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 50 mM ethylenediaminetetraacetic acid, 50 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 1% Triton X-100) and sonicated for 15 sec at 40 V over ice. The solution was centrifuged at 1,600g for 10 min at 4°C, and the supernatant was subjected to an assay kit from Cayman Chemical (Ann Arbor, MI).
Real-Time Polymerase Chain Reaction Analysis
Total RNA was extracted from tissue samples after 24 hr reperfusion using TRIzol reagent (Invitrogen, Carlsbad, CA). Complementary DNA was synthesized from 3.5 µg of total RNA using Oligo (dT)12–18 primer (Invitrogen) and murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA). The expressions of target mRNAs were detected by quantitative real-time polymerase chain reaction (7300 Real Time PCR System, Applied Biosystems) with SYBR Green as detection dye. Primer sequences are shown in Table S1, SDC, http://links.lww.com/TP/A985. Relative expression of each mRNA was calculated using ΔΔCt threshold method. The level of β-actin mRNA was used for normalization. Relative expression of mRNA was expressed as fold change in comparison to the control group.
Mitochondrial DNA Copy Number
DNA was extracted from 20 mg of whole kidney tissue using the DNeasy kit from Qiagen (Valencia, CA). The concentration of DNA was determined using the Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). Five nanograms of DNA was used for real-time polymerase chain reaction using primers specific for the mitochondrial gene, Nicotinamide adenine dinucleotide dehydrogenase subunit 6 (ND-6). Primers for the gene POU class 5 homeobox 1 (Pou5f1) were used as genomic DNA control (35). Primer sequences are shown in Table S1, SDC, http://links.lww.com/TP/A985. Relative quantification of mitochondrial DNA copies to genomic DNA was calculated using ΔΔCt threshold method.
Whole kidney protein levels were examined by Western blotting. Briefly, 100 mg of pulverized tissue was homogenized in 500 μL of lysis buffer (10 mM Tris-HCl pH 7.5, 120 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) containing a protease inhibitor cocktail (Roche Diagnostics) using high-frequency sonication. Samples were then centrifuged at 12,000 rpm for 15 min at 4°C, and supernatant was collected for further analyses. Protein concentration was subsequently determined by a BIO-RAD DC protein assay kit (BIO-RAD, Hercules, CA). Fifty micrograms of protein was fractionated on a Bis-Tris gel and transferred to a nitrocellulose membrane. The membranes were blocked by incubation in 0.2× phosphate-buffered saline with 0.1% casein and incubated with primary anti-SDH subunit B, PGC1α, mtTFA, IκB-α, p-NF-κB (ser 276), NF-κB, iNOS, nitrotyrosine and β-actin antibodies (Santa Cruz Biotechnology) in 0.2× phosphate-buffered saline with 0.1% casein and 0.1% Tween 20. After washing, the blots were incubated subsequently with corresponding fluorescent secondary antibody (LI-COR, Lincoln, NE). Bands were detected using the Odyssey FC Dual-Mode Imaging system 2800 (LI-COR). Protein density was measured using NIH ImageJ software.
Immortalized human kidney cells (HK-2) (51) were cultured in Dulbecco’s minimum essential medium (DMEM) (Invitrogen) containing 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin and 2 mM glutamine and maintained in a 37°C incubator with 5% CO2. Cells were seeded on a cell culture plate overnight and then treated with 0.1% dimethyl sulfoxide or 250 nM of SRT1720 for 24 hr.
Measurement of Mitochondrial Mass
Cells were incubated with DMEM containing 100 nM of MitoTracker green FM (Life Technologies, Eugene, OR) for 30 min. Next, cells were trypsinized, centrifuged, resuspended in DMEM, and immediately subjected to flow cytometry (FACSverse, BD biosciences, San Jose, CA). Live cells were gated by forward and side scatter and negative staining for propidium iodide. Data were analyzed using the FlowJO 7.6.5 software.
Data are expressed as mean±standard error and compared by one-way analysis of variance using Student-Newman-Keuls’ test. Differences in values were considered significant at P less than 0.05. All experiments were performed with n=4 to 5 per group except for Western blotting n=3-4/group. In vitro experiments were performed with n=4 per group.
1. Powell JT, Tsapepas DS, Martin ST, et al. Managing renal transplant ischemia reperfusion injury: novel therapies in the pipeline. Clin Transplant
2013; 27: 484.
2. Goto R, Issa F, Heidt S, et al. Ischemia-reperfusion injury accelerates human antibody-mediated transplant vasculopathy. Transplantation
2013; 96: 139.
3. Aydin Z, van Zonneveld AJ, de Fijter JW, et al. New horizons in prevention and treatment of ischaemic injury to kidney transplants. Nephrol Dial Transplant
2007; 22: 342.
4. Sharfuddin AA, Molitoris BA. Pathophysiology of ischemic acute kidney injury. Nat Rev Nephrol
2011; 7: 189.
5. Plotnikov EY, Kazachenko AV, Vyssokikh MY, et al. The role of mitochondria in oxidative and nitrosative stress during ischemia/reperfusion in the rat kidney. Kidney Int
2007; 72: 1493.
6. Saba H, Batinic-Haberle I, Munusamy S, et al. Manganese porphyrin reduces renal injury and mitochondrial damage during ischemia/reperfusion. Free Radic Biol Med
2007; 42: 1571.
7. Rasbach KA, Schnellmann RG. PGC-1alpha over-expression promotes recovery from mitochondrial dysfunction and cell injury. Biochem Biophys Res Commun
2007; 355: 734.
8. Lin SJ, Defossez PA, Guarente L. Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae
2000; 289: 2126.
9. Tissenbaum HA, Guarente L. Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans
2001; 410: 227.
10. Rogina B, Helfand SL. Sir2 mediates longevity in the fly through a pathway related to calorie restriction. Proc Natl Acad Sci U S A
2004; 101: 15998.
11. Boily G, Seifert EL, Bevilacqua L, et al. SirT1 regulates energy metabolism
and response to caloric restriction in mice. PLoS One
2008; 3: e1759.
12. Afshar G, Murnane JP. Characterization of a human gene with sequence homology to Saccharomyces cerevisiae
1999; 234: 161.
13. Imai S-i, Armstrong CM, Kaeberlein M, et al. Transcriptional silencing and longevity protein Sir2 is an NAD-dependenthistone deacetylase. Nature
2000; 403: 795.
14. Xie J, Zhang X, Zhang L. Negative regulation of inflammation by SIRT1. Pharmacol Res
2013; 67: 60.
15. Yeung F, Hoberg JE, Ramsey CS, et al. Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase. EMBO J
2004; 23: 2369.
16. Luo J, Nikolaev AY, Imai S, et al. Negative control of p53 by Sir2alpha promotes cell survival under stress. Cell
2001; 107: 137.
17. Fan H, Yang HC, You L, et al. The histone deacetylase, SIRT1, contributes to the resistance of young mice to ischemia/reperfusion-induced acute kidney injury. Kidney Int
2013; 83: 404.
18. Rodgers JT, Lerin C, Haas W, et al. Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. Nature
2005; 434: 113.
19. Rodgers JT, Lerin C, Gerhart-Hines Z, et al. Metabolic adaptations through the PGC-1 alpha and SIRT1 pathways. FEBS Lett
2008; 582: 46.
20. Nogueiras R, Habegger KM, Chaudhary N, et al. Sirtuin 1
and sirtuin 3: physiological modulators of metabolism. Physiol Rev
2012; 92: 1479.
21. He W, Wang Y, Zhang MZ, et al. Sirt1 activation protects the mouse renal medulla from oxidative injury. J Clin Invest
2010; 120: 1056.
22. Hasegawa K, Wakino S, Yoshioka K, et al. Sirt1 protects against oxidative stress
-induced renal tubular cell apoptosis by the bidirectional regulation of catalase expression. Biochem Biophys Res Commun
2008; 372: 51.
23. Hasegawa K, Wakino S, Yoshioka K, et al. Kidney-specific overexpression of Sirt1 protects against acute kidney injury by retaining peroxisome function. J Biol Chem
2010; 285: 13045.
24. Shah KG, Rajan D, Jacob A, et al. Attenuation of renal ischemia and reperfusion injury by human adrenomedullin and its binding protein. J Surg Res
2010; 163: 110.
25. Milne JC, Lambert PD, Schenk S, et al. Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes. Nature
2007; 450: 712.
26. Chatterjee PK, Patel NS, Sivarajah A, et al. GW274150, a potent and highly selective inhibitor of iNOS, reduces experimental renal ischemia/reperfusion injury. Kidney Int
2003; 63: 853.
27. Funk JA, Odejinmi S, Schnellmann RG. SRT1720 induces mitochondrial biogenesis
and rescues mitochondrial function after oxidant injury in renal proximal tubule cells. J Pharmacol Exp Ther
2010; 333: 593.
28. Price NL, Gomes AP, Ling AJ, et al. SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function. Cell Metab
2012; 15: 675.
29. Wu Z, Puigserver P, Andersson U, et al. Mechanisms controlling mitochondrial biogenesis
and respiration through the thermogenic coactivator PGC-1. Cell
1999; 98: 115.
30. Armstrong D, Browne R. The analysis of free radicals, lipid peroxides, antioxidant enzymes and compounds related to oxidative stress
as applied to the clinical chemistry laboratory. Adv Exp Med Biol
1994; 366: 43.
31. Yagi K. Simple assay for the level of total lipid peroxides in serum or plasma. Methods Mol Biol
1998; 108: 101.
32. Czubryt MP, McAnally J, Fishman GI, et al. Regulation of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 alpha) and mitochondrial function by MEF2 and HDAC5. Proc Natl Acad Sci U S A
2003; 100: 1711.
33. Lagouge M, Argmann C, Gerhart-Hines Z, et al. Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Cell
2006; 127: 1109.
34. Gerhart-Hines Z, Rodgers JT, Bare O, et al. Metabolic control of muscle mitochondrial function and fatty acid oxidation through SIRT1/PGC-1alpha. EMBO J
2007; 26: 1913.
35. Chau CM, Evans MJ, Scarpulla RC. Nuclear respiratory factor 1 activation sites in genes encoding the gamma-subunit of ATP synthase, eukaryotic initiation factor 2 alpha, and tyrosine aminotransferase. Specific interaction of purified NRF-1 with multiple target genes. J Biol Chem
1992; 267: 6999.
36. Evans MJ, Scarpulla RC. NRF-1: a trans-activator of nuclear-encoded respiratory genes in animal cells. Genes Dev
1990; 4: 1023.
34. Virbasius JV, Scarpulla RC. Activation of the human mitochondrial transcription factor A gene by nuclear respiratory factors: a potential regulatory link between nuclear and mitochondrial gene expression in organelle biogenesis. Proc Natl Acad Sci U S A
1994; 91: 1309.
38. Antoshechkin I, Bogenhagen DF, Mastrangelo IA. The HMG-box mitochondrial transcription factor xl-mtTFA binds DNA as a tetramer to activate bidirectional transcription. EMBO J
1997; 16: 3198.
39. Montoya J, Perez-Martos A, Garstka HL, et al. Regulation of mitochondrial transcription by mitochondrial transcription factor A. Mol Cell Biochem
1997; 174: 227.
40. Inagaki H, Kitano S, Lin KH, et al. Inhibition of mitochondrial gene expression by antisense RNA of mitochondrial transcription factor A (mtTFA). Biochem Mol Biol Int
1998; 45: 567.
41. Kong X, Wang R, Xue Y, et al. Sirtuin 3, a new target of PGC-1alpha, plays an important role in the suppression of ROS and mitochondrial biogenesis
. PLoS One
2010; 5: e11707.
42. Goligorsky MS, Brodsky SV, Noiri E. Nitric oxide in acute renal failure: NOS versus NOS. Kidney Int
2002; 61: 855.
43. Ling H, Gengaro PE, Edelstein CL, et al. Effect of hypoxia on proximal tubules isolated from nitric oxide synthase knockout mice. Kidney Int
1998; 53: 1642.
44. Noiri E, Nakao A, Uchida K, et al. Oxidative and nitrosative stress in acute renal ischemia. Am J Physiol Renal Physiol
2001; 281: F948.
45. Qi S, Xu D, Ma A, et al. Effect of a novel inducible nitric oxide synthase inhibitor, FR260330, in prevention of renal ischemia/reperfusion injury in vervet monkeys. Transplantation
2006; 81: 627.
46. Cruthirds DL, Novak L, Akhi KM, et al. Mitochondrial targets of oxidative stress
during renal ischemia/reperfusion. Arch Biochem Biophys
2003; 412: 27.
47. Beckman JS, Beckman TW, Chen J, et al. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A
1990; 87: 1620.
48. Kleinert H, Pautz A, Linker K, et al. Regulation of the expression of inducible nitric oxide synthase. Eur J Pharmacol
2004; 500: 255.
49. Kinsey GR, Li L, Okusa MD. Inflammation in acute kidney injury. Nephron Exp Nephrol
2008; 109: e102.
50. Kelly KJ, Williams WW Jr, Colvin RB, et al. Intercellular adhesion molecule-1-deficient mice are protected against ischemic renal injury. J Clin Invest
1996; 97: 1056.
51. Ryan MJ, Johnson G, Kirk J, et al. HK-2: an immortalized proximal tubule epithelial cell line from normal adult human kidney. Kidney Int
1994; 45: 48.