1521-0103/354/2/142–151$25.00 http://dx.doi.org/10.1124/jpet.115.224386THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 354:142–151, August 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Activation of Sirtuin-1 Promotes Renal Fibroblast Activation andAggravates Renal Fibrogenesis s

Murugavel Ponnusamy, Michelle A. Zhuang, Xiaoxu Zhou, Evelyn Tolbert, George Bayliss,Ting C. Zhao, and Shougang ZhuangDepartment of Medicine, Rhode Island Hospital and Alpert Medical School, Brown University, Providence, Rhode Island (M.P.,M.A.Z., X.Z., E.T., G.B., S.Z.); Department of Surgery, Roger William Medical Center, Boston University Medical School,Providence, Rhode Island (T.C.Z.); and Department of Nephrology, Shanghai East Hospital, Tongji University School ofMedicine, Shanghai, China (S.Z.)

Received March 12, 2015; accepted May 27, 2015

ABSTRACTAlthough activation of sirtuin-1 (SIRT1) has been shown toprotect the kidney from acute injury, its role in renal fibrosisremains controversial since both inhibition and activation ofSIRT1 have been reported to attenuate renal fibrosis. To resolvethis conflict, we further examined the effect of SIRT1 activatorson the activation of renal interstitial fibroblasts and developmentof renal fibrosis in vivo and in vitro. In a murine model of renalfibrosis induced by unilateral ureteral obstruction, administrationof SRT1720 (N-[2-[3-(piperazin-1-ylmethyl)imidazo[2,1-b][1,3]thiazol-6-yl]phenyl]quinoxaline-2-carboxamide), a potent acti-vator of SIRT1, accelerated deposition of collagen fibrils andincreased expression of fibroblast activation markers (a-smoothmuscle actin [a-SMA], collagen I, and fibronectin) in theobstructive kidney of mice. In cultured rat renal interstitialfibroblasts (NRK-49F), exposure of cells to SRT1720 or YK-3-237(B-[2-methoxy-5-[(1E)-3-oxo-3-(3,4,5-trimethoxyphenyl)-1-propen-

1-yl]phenyl]-boronic acid), another SIRT1 activator, also resultedin enhanced expression of a-SMA and fibronectin. Mechanisticstudies showed that augmentation of renal fibrogenesis bySRT1720 is associated with elevated phosphorylation ofepidermal growth factor receptor (EGFR) and platelet-derivedgrowth factor receptor b (PDGFRb). SRT1720 treatment alsoincreased the phosphorylation of signal transducer and activatorof transcription 3 and protein kinase B in the fibrotic kidney andNRK-49F cells. However, SRT1720 treatment did not affectexpression of proliferating cell nuclear protein, a proliferationmarker and activation of extracellular signal regulated kinase 1/2in vitro and in vivo. These results indicate that SIRT1-activatingcompounds can provoke renal fibrogenesis through amechanisminvolved in the activation of EGFRand PDGFR signaling pathwaysand suggest that long-term use of SIRT1 activators risks thedevelopment and progression of chronic kidney disease.

IntroductionTubulointerstitial fibrosis is a common event for the pro-

gression of chronic kidney disease regardless of the primarycauses of renal disease (Tampe and Zeisberg, 2014). Renalfibrogenesis is characterized by activation of renal interstitialfibroblasts and subsequential production of an excessiveamount of extracellular matrix (ECM) proteins. The trans-formation of fibroblasts into myofibroblasts is a core event forthe development of fibrotic lesions in the kidney (Zeisberg and

Neilson, 2010). Transforming growth factor (TGF)-b1 and othergrowth factors, such as epidermal and platelet-derived growthfactors, are critically involved in the fibrotic process (Bonner,2004; Liu et al., 2012, 2013). These cytokines/growth factorstrigger their cellular events through binding to their receptorsand subsequent activation of multiple downstream signalingpathways, such as signal transducer and activator of tran-scription 3 (STAT3), protein kinase B (AKT), and extracellularsignal-regulated kinase 1/2 (ERK1/2) (Qin and Han, 2010; Liuet al., 2012, 2013; Kitada et al., 2013; Ponnusamy et al., 2013).It is evident that activation of epidermal growth factorreceptors (EGFRs) and platelet-derived growth factor receptors(PDGFRs) contributes to activation of renal interstitialfibroblasts and development of renal fibrosis (Bonner, 2004;LeBleu and Kalluri, 2011; Liu et al., 2011, 2012).Epigenetic modifications, such as acetylation/deacetylation,

have also been linked to the pathogenesis of chronic kidney

This work was supported by the National Institutes of Health NationalInstitute of Diabetes and Digestive and Kidney Diseases [Grant 5R01-DK085065]; the National Nature Science Foundation of China [Grants81270778 and 81470920]; and the Key Discipline Construction Project of thePudong Health Bureau of Shanghai [Grant PWZ2014-06].

dx.doi.org/10.1124/jpet.115.224386.s This article has supplemental material available at jpet.aspetjournals.org.

ABBREVIATIONS: Ac-H3K9, acetylation of histone H3 at lysine 9; AKT, protein kinase B; ECM, extracellular matrix; EGFR, epidermal growth factorreceptor; ERK1/2, extracellular signal-regulated kinase 1/2; EX527, 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide; HDAC, histonedeacetylase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PCNA, proliferating cell nuclear antigen; PDGFR, platelet-derivedgrowth factor receptor; SIRT1, sirtuin-1; SRT1720, N-[2-[3-(piperazin-1-ylmethyl)imidazo[2,1-b][1,3]thiazol-6-yl]phenyl]quinoxaline-2-carboxamide;a-SMA, a-smooth muscle actin; SOCS, suppressor of cytokine signaling; STAT3, signal transducer and activator of transcription 3; TGF,transforming growth factor; UUO, unilateral ureteral obstruction; YK-3-237, B-[2-methoxy-5-[(1E)-3-oxo-3-(3,4,5-trimethoxyphenyl)-1-propen-1-yl]phenyl]-boronic acid.

142

http://jpet.aspetjournals.org/content/suppl/2015/05/28/jpet.115.224386.DC1Supplemental material to this article can be found at:

at ASPE

T Journals on A

ugust 11, 2020jpet.aspetjournals.org

Dow

nloaded from

disease (Liu et al., 2013; Van Beneden et al., 2013; Tampe andZeisberg, 2014). Protein acetylation is regulated by a networkof enzyme systems, namely, histone acetyltransferases andhistone deacetylases (HDACs). HDACs can modulate bothacetylation of both histone and nonhistone proteins associatedwith fibrogenic process, such as STAT3 (Rombouts et al., 2002;Pang and Zhuang, 2010; Qin and Han, 2010). Among the fourclasses of HDACs, class I and II HDACs have been extensivelystudied for their role in tissue fibrogenesis (Pang et al., 2009,2011; Pang and Zhuang, 2010). It is evident that smallmolecular inhibitors acting on these two classes of HDACswere effective in attenuating fibrosis in multiple organsincluding kidney (Pang et al., 2009; Liu et al., 2013; VanBeneden et al., 2013). Apart from class I and IIHDACs, the roleof sirtuins (SIRTs), a class III HDAC, has also been intensivelystudied in animal models of kidney diseases. Among the sevenmembers of SIRTs, SIRT1 is ubiquitously expressed in renaltissue, and has been shown to be implicated in renal protectionunder ischemic, hypoxic, and calorie-restricted conditions (Fanet al., 2013; Kitada et al., 2013; Dong et al., 2014). However, therole of SIRT1 in renal fibrogenesis remains controversial eventhough its activity increases in the fibrotic kidney (Ponnusamyet al., 2014). Earlier studies have indicated that activation ofSIRT1 by resveratrol, a polyphenol found in red wine, canalleviate renal fibrosis induced by unilateral ureteral obstruc-tion (UUO) inmice (Li et al., 2010). However, our recent studiesshowed that inhibition of SIRT1 and 2 with sirtinol attenuatedrenal fibrosis in the samemodel. In addition, specific inhibitionof SIRT1 by EX527 (6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) or silencing of SIRT1 with siRNA suppressedactivation of renal interstitial fibroblasts (Ponnusamy et al.,2014).These obviously conflicting results make it difficult toexplain the functionality of SIRT1 in renal fibrogenesis and raisethe issue of whether the antifibrotic effect of resveratrol is due toits nonspecific effects. It is well documented that this compoundindeed has off-target effects (Pacholec et al., 2010). Indeed,a recent report from Venturelli et al. (2013) showed thatresveratrol has an inhibitory effect on class I, II, and IV HDACs(Venturelli et al., 2013). With the view that blocking of class I/IIHDACs can attenuate renal fibrosis (Pang et al., 2009; Liu et al.,2013), this suggests the possibility that resveratrol may alleviaterenal fibrosis through a mechanism involved in the inhibition ofclass I/II HDACs rather than activation of SIRT1. Therefore,additional experiments are needed to characterize the role ofSIRT1 activation in regulating renal fibrosis by using morespecific SIRT1 pharmacological activators.Efforts have been made to discover new molecules that are

able to stimulate SIRT1 activities more specifically andpotentially than resveratrol. SRT1720 (N-[2-[3-(piperazin-1-ylmethyl)imidazo[2,1-b][1,3]thiazol-6-yl]phenyl]quinoxaline-2-carboxamide) is structurally unrelated to resveratrol anddoes not share the off-target effects of resveratrol (Villalbaand Alcain, 2012); therefore, it is a useful tool for verifyingputative SIRT1-dependent effects in vivo. In this study, weinvestigated that the influence of SRT1720 on the progressionof renal fibrosis in a murine model of renal fibrosis induced byUUO and on the activation of cultured renal fibroblasts.

Materials and MethodsChemicals and Antibodies. Antibodies to fibronectin, collagen I(A2),

EGFR, glyceraldehyde-3-phosphate dehydrogenase, and proliferating cell

nuclear antigen (PCNA) were obtained from Santa Cruz Biotechnology,Inc. (Santa Cruz, CA). All other antibodies used in this study werepurchased from Cell Signaling Technology (Danvers, MA). SRT1720was purchased from EMD Millipore (Billerica, MA). a-Smooth muscleactin (a-SMA) and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Cell Culture and Treatments. Rat renal interstitial fibroblasts(NRK-49F) were cultured in Dulbecco’s modified Eagle’s mediumwithF12 containing 5% fetal bovine serum and 0.5% penicillin andstreptomycin in an atmosphere of 5% CO2 and 95% air at 37°C. Toreduce the interference of growth factors in serum with SRT1720activity, NRK-49F cells were grown in Dulbecco’s modified Eagle’smedium with F12 with 2.5% fetal bovine serum for 24 hours, andSRT1720 was directly added to subconfluent NRK-49F cells andincubated for 36 hours to determine the effects of SRT1720 on renalfibroblast activation.

Animals and Experimental Design. The UUO model wasestablished in 6–8 week old male C57 black mice that weighed 20–25 g(The Jackson Laboratory, Bar Harbor, ME) as described in previousstudies (Pang et al., 2009, 2010). Briefly, the abdominal cavity wasexposed via a midline incision and the left ureter was isolated andligated. The contralateral kidney was used as a control. To examine theeffects of SRT1720 on renal fibrosis after UUO injury, SRT1720 at 200mg/kg body weight (prepared in 50 ml of dimethylsulfoxide) wasintraperitoneally administered immediately after ureteral ligation andthen given daily for 4 days. The dose of SRT1720 was selected based ona previous report (Imanishi et al., 2012). Control mice were injectedwith an equal volume of dimethylsulfoxide. The animals were sacrificedand the kidneys were collected at day 5 for protein analysis andhistologic examination. All experimental procedures were performedaccording to the US National Institutes of Health Guidelines for theCare and Use of Laboratory Animals. The protocol (#0135-13) wasapproved by the Lifespan Animal Welfare Committee at Rhode IslandHospital. All surgery was performed under sodium pentobarbitalanesthesia, and effort was made to minimize suffering.

Masson Trichrome Staining. For assessment of renal fibrosis,Masson trichrome staining was performed according to the protocolprovided by the manufacturer (Sigma-Aldrich). The semiquantitativeanalysis of the collagen tissue area (blue-colored area) was measuredusing ImageJ software developed at the National Institutes of Health(Bethesda, MD). The positive staining area from each microscopicfield (200�) was calculated and graphed.

Immunoblot Analysis. To prepare protein samples for westernblotting, the kidney tissue samples were homogenized in the cell lysisbuffer (Cell Signaling Technology) with protease inhibitor cocktail(Roche, Basel, Switzerland). After various treatments, cells werewashed once with ice-cold phosphate-buffered saline and harvested ina cell lysis buffer mixed with a protease inhibitor cocktail. The proteinlevel was measured by the bicinchoninic acid method and proteinswere separated by SDS-PAGE and transferred to nitrocellulosemembranes. After incubation with 5% nonfat milk for 1 hour at roomtemperature, the membranes were incubated with primary antibodyovernight at 4°C and washed with Tris buffered saline with Tween-20.Then, the membranes were incubated with appropriate horseradishperoxidase–conjugated secondary antibody for 1 hour in roomtemperature. After washing the membrane with Tris buffered salinewith Tween-20, bound antibodies were visualized by chemilumines-cence detection using electrochemiluminescence solution obtainedfrom GE Healthcare Life Sciences (Pittsburgh, PA).

Densitometry Analysis. The semiquantitative analyses of dif-ferent proteins were carried out by using the ImageJ software.Quantification is based on the intensity (density) of the band, which iscalculated by the area and pixel value of the band. The quantificationdata are given as the ratio between the target protein and loadingcontrol (housekeeping protein) and the ratio between the phosphor-ylated protein and corresponding total protein.

Statistical Analysis. Data are presented as mean 6 S.D. andwere subjected to one-way analysis of variance. Multiple means were

Sirt1 Activation Aggravates Renal Fibrosis 143

at ASPE

T Journals on A

ugust 11, 2020jpet.aspetjournals.org

Dow

nloaded from

compared using Tukey’s test, and the differences between two groupswere determined by Student’s t test. P , 0.01 was consideredstatistically significant.

ResultsSRT1720 Enhances Deposition of ECM Components

and Activation of Renal Fibroblasts in the Kidney afterUUO Injury. Progressive interstitial fibrosis is the result ofexcessive production of ECM components by activatedfibroblasts (Zeisberg and Neilson, 2010). Ponnusamy et al.(2014) have shown that SIRT1 and 2 activities are increasedduring renal fibrosis induced by UUO injury and treatmentwith SIRT1/2 selective inhibitors can attenuate deposition ofECM proteins and inhibited activation of renal interstitialfibroblasts in the mouse model of renal fibrosis induced byUUO, suggesting that SIRT1/2 are critical regulators of renalfibrogenesis. If this conclusion is correct, SIRT1 activationwould exert an opposite effect by enhancing renal fibrosis. Totest this hypothesis, we collected the kidney after 4 days ofUUO injury with or without SRT1720 treatment and thenexamined the effect of SRT1720 on renal fibrosis. Massontrichrome staining illustrates that the deposition and accumu-lation of ECM components were increased in the tubulointer-stitial space as a consequence of myofibroblast activation, andadministration of SRT1720 further increased the deposition ofECM components in the interstitial space (Fig. 1A). Semi-quantitative analysis of Masson trichrome–positive areasrevealed a 4-fold increase of ECM components in the obstructive

kidney compared with the sham kidney. SRT1720 treatmentincreased ECM deposition by more than 3-fold compared withUUO injury alone (Fig. 1B). Immunoblot analysis of wholekidney tissue lysate indicated that acetylation of histone H3 atlysine 9 (Ac-H3K9) was increased in the injured kidney and itslevel was significantly decreased in the kidney of mice treatedwith SRT1720, indicating that the dose of SRT1720 waseffective in elevating renal SIRT1 deacetylase activity (Fig. 1, Cand D). In addition, there was also an increase in theexpression of total H3 in UUO-injured kidney; however, itslevel was not affected by SRT1720 treatment (Fig. 1, C and E).Immunoblot analysis showed that UUO injury increased theexpression of SIRT1 but that SRT1720 did not reduce itsexpression (Supplemental Fig. 1).The expansion of renal interstitial fibrosis is classically

manifested by an increase in the population of myofibroblasts,i.e., the phenotypically transformed fibroblasts that expressa-SMA and produce ECM components (Meran and Steadman,2011). By immunoblot analysis, we examined the expression offibroblast activation and proliferation markers in UUO-injuredkidney of mice treated with or without SRT1720. As shown inFig. 2, increased expression of fibroblast activation markers(a-SMA, collagen I, and fibronectin), as well as proliferationmarkers (PCNA), were observed in the obstructed kidney.Administration of SRT1720 profoundly increased (about 2-fold)the expression levels of a-SMA, collagen I, and fibronectin (Fig.2, A–D); however, PCNA expression was not affected by thiscompound (Fig. 2, E and F). Thus, these data indicate thatSRT1720 can induce aggressive activation of fibroblasts and

Fig. 1. SRT1720 enhances the deposition of ECMand development of fibrosis in obstructed kidneys.(A) Photomicrographs illustrating Masson trichromestaining of kidney tissue after treatment with orwithout SRT1720. (B) The Masson trichrome–positivetubulointerstitial area (blue) relative to the whole areafrom 10 random cortical fields (200�) (mean 6 S.D.)was analyzed. Data are represented as the mean6S.D. Means with different lowercase letters aresignificantly different from one another (P , 0.01).(C) Kidney tissue lysates were subjected to immuno-blot analysis with antibodies for acetyl-H3K9 (Ac-H3K9), total H3, or b-actin. The levels of Ac-H3K9and total H3 were quantified by densitometry andnormalized with b-actin (D and E). Values are mean 6S.D. (n = 6). Bars with different lowercase letters (a–c)are significantly different from one another (P , 0.01).

144 Ponnusamy et al.

at ASPE

T Journals on A

ugust 11, 2020jpet.aspetjournals.org

Dow

nloaded from

accumulation of ECM but not affect proliferation of interstitialfibroblast cells in the kidney after UUO injury.Treatment with SIRT1 Activators Potentiates Cul-

tured Renal Interstitial Fibroblast Activation. To con-firm the role of SIRT1 in mediating renal fibroblast activationin the mousemodel, we examined the effect of SRT1720 on theexpression of fibrogenic markers in cultured renal interstitialfibroblast cells (NRK-49F). NRK-49F grown in reduced levelsof serum (2.5% fetal bovine serum) was exposed to variousconcentrations (0.5–2 mM) of SRT1720 for 36 hours, and theexpression levels of the fibroblast activation markers (a-SMAand fibronectin) were assessed. As shown in Fig. 3A, SRT1720dose dependently increased expression of those proteins.Densitometry analysis demonstrated that SRT1720 increasedthe expression of a-SMA (Fig. 3B) and fibronectin (Fig. 3C) byapproximately 2- to 3-fold at a dose of 1 mM, and their levelswere further enhanced in fibroblasts exposed to 2 mM ofSRT1720. To demonstrate that this increase is due to theactivation of SIRT1 deacetylase activity, we also examinedthe level of Ac-H3K9. As expected, SRT1720 treatmentdecreased the level of Ac-H3K9 in a dose-dependent manner(0.5–2 mM). The level of Ac-H3K9 was significantly decreased(∼70%) at a concentration of 1 mM SRT1720 treatment andfurther decreased more than 90% at a dose of 2 mM (Fig. 3, Aand D). However, SRT1720 treatment did not increase thelevel of PCNA (Fig. 3, E and F) and cell proliferation, even atthe maximum concentration (2 mM), as indicated by cellcounting (Fig. 3G) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Fig. 3H). Similarly,SRT1720 treatment did not increase the PCNA level incultured renal proximal tubular cells (data not shown). Thesedata are consistent with our in vivo data, indicating that

SRT1720 triggers activation but not proliferation of renalinterstitial fibroblasts.We further examined the effect of YK-3-237 (B-[2-methoxy-

5-[(1E)-3-oxo-3-(3,4,5-trimethoxyphenyl)-1-propen-1-yl]phenyl]-boronic acid), another potent activator of Sirt1 (Yi et al., 2013),on the activation of renal interstitial fibroblasts using NRK-49Fcells. Our data demonstrated that exposure of cells to YK-3-237also significantly reduced expression of a-SMA and fibro-nectin in a dose-dependent manner, with the maximum inhibi-tion occurring at 10 mM. This dose of the inhibitor completelyblocked expression of Ac-H3K9, suggesting its effectivenessin the activation of Sirt1 (Supplemental Fig. 2). Collec-tively, our data provide strong evidence that SIRT1 playsa critical role in mediating activation of renal interstitialfibroblasts.SRT1720 Enhances UUO-Induced Phosphorylation

of EGFR and PDGFRb in Obstructed Kidneys. Activa-tion of growth factor signaling pathways is involved in theregulation of fibrosis development. Previous studies haveshown that EGFR and PDGFRb are two major contributors torenal fibroblast activation and renal fibrogenesis (Ludewiget al., 2000; Terzi et al., 2000; Bonner, 2004; Di Pascoli et al.,2013). To determine whether SRT1720 exerts its profibroticeffects through activation of these two receptors, we examinedthe effect of this agent on the phosphorylation of EGFR atTyr1068 (Y1068) and PDGFRb at Tyr751 (Y751). As shown inFig. 4, the level of phospho-EGFR was increased by ∼4-foldafter 4 days in the kidney with UUO injury, and SRT1720treatment enhanced phospho-EGFR level up to 4-fold whencompared with the UUO-injured kidney treated with thevehicle. Interestingly, SRT1720 administration also increasedphospho-EGFR levels more than 8-fold in the sham-operated

Fig. 2. Administration of SRT1720 increases renalinterstitial fibroblast activation in obstructed kid-neys. Kidney tissue lysates were subjected to immu-noblot analysis with antibodies against a-SMA,collagen I, fibronectin, PCNA, or b-actin (A and E).Representative immunoblots from three independentexperiments are shown. The levels of a-SMA,collagen I, fibronectin, and PCNA were quantifiedby densitometry and normalized with b-actin (B–Dand F). Values are mean 6 S.D. (n = 6). Bars withdifferent lowercase letters (a–c) are significantlydifferent from one another (P , 0.01).

Sirt1 Activation Aggravates Renal Fibrosis 145

at ASPE

T Journals on A

ugust 11, 2020jpet.aspetjournals.org

Dow

nloaded from

kidney compared with the control kidney (Fig. 4, A and B). Inaddition, UUO injury resulted in increased expression of totalEGFR; however, SRT1720 treatment did not alter itsexpression levels (Fig. 4, A and C). Similarly, SRT1720 wasalso effective in potentiating PDGFRb phosphorylation inboth sham-operated and UUO-injured kidneys (Fig. 4, A andD); however, it was not effective in altering the level of totalPDGFRb (Fig. 4, A and E). Collectively, our data suggest thatSRT1720 treatment enhances phosphorylation of both EGFRand PDGFRb in the fibrotic kidney.SRT1720 Enhances Phosphorylation/Activation of

EGFR and PDGFRb in Cultured Renal Fibroblasts. Tospecifically demonstrate that SRT1720-induced enhancement ofEGFRandPDGFRb activation occurs in renal fibroblasts duringUUO injury, we further examined the effect of SRT1720 on theirphosphorylation status in cultured renal fibroblasts. Consistent

with our in vivo data, exposure to SRT1720 also dose-dependentlyincreased the phosphorylation level of EGFR and PDGFRb incultured NRK-49F cells (Fig. 5, A and B). In addition, SRT1720increased the level of total EGFR, with the maximum effectoccurring at 2 mM; however, this agent did not affect the level oftotal PDGFRb (Fig. 5, A and C). Thus, SRT1720 can enhanceEGFRandPDGFRb phosphorylation in renal fibroblasts butwithdifferent effects on their total protein levels.SRT1720 Enhances Phosphorylation of STAT3, but

Not ERK1/2 in UUO-Injured Kidney and CulturedRenal Fibroblasts. EGFR and PDGFRb exert their biologicfunctions through activation of several intracellular signalingpathways including STAT3, AKT, and ERK1/2 (Ludewig et al.,2000; Liu et al., 2012; Tang et al., 2013), and these pathwaysare also associated with development of renal fibrosis (Panget al., 2010; Lan and Du, 2015). Thus, we further examined the

Fig. 3. SRT1720 treatment enhances activation ofcultured renal interstitial fibroblasts. NRK-49F cellswere cultured in 2.5% fetal bovine serum containingmedium and incubated with different concentrations ofSRT1720 (0–2 mM) for 36 hours. Then, cell lysates wereprepared and subjected to immunoblot analysis withantibodies against a-SMA, fibronectin, Ac-H3K9, PCNA,or glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(A and E). Representative immunoblots from threeindependent experiments are shown. The levels of Ac-H3K9, a-SMA, fibronectin, and PCNA were quantifiedby densitometry and normalized with GAPDH (B–D andF). NRK-49F cells were treated with the indicatedconcentration of SRT1720 for 36 hours and cells wererandomly photographed in bright field (200�). Cellproliferation was measured by cell counting (G), or theMTT assay (H). Values are mean 6 S.D. of threeindependent experiments. Bars with different lowercaseletters (a–d) are significantly different from one another(P , 0.01).

146 Ponnusamy et al.

at ASPE

T Journals on A

ugust 11, 2020jpet.aspetjournals.org

Dow

nloaded from

influence of SRT1720 on their phosphorylation in vivo and invitro. As shown in Fig. 6, A–C, the phosphorylation levels ofSTAT3 (Y705) and total STAT3 were increased in theobstructed kidneys. Interestingly, SRT1720 administrationfurther increased the levels of both phosphorylated and totalSTAT3 inUUO-injured and sham-operated kidneys. Comparedwith the sham-operated kidney, the UUO-injured kidney alsoshowed an increase in the phosphorylation of AKT (S473) and

SRT1720 treatment enhanced UUO-induced AKT phosphory-lation as clearly indicated by the ratio of phospho-AKT/AKT. Insharp contrast to elevated phospho-AKT, administration ofSRT1720 resulted in reduction in the level of AKT in bothsham-operated and UUO-injured kidneys (Fig. 6, A, D, and E).Although increased levels of phosphorylated and total ERK1/2were detected in the obstructed kidney, treatment withSRT1720 did not affect these responses (Fig. 6, F and G).

Fig. 4. SRT1720 increases phosphorylation of EGFRand PDGFRb in obstructed kidneys. Kidney tissue ly-sates were prepared and subjected to immunoblot anal-ysis with antibodies against phospho-EGFR (Tyr1068),EGFR, phospho-PDGFRb (Tyr751), PDGFRb, orb-actin (A). The phospho-EGFR, phospho-PDGFRb,EGFR, PDGFRb, and b-actin were quantified bydensitometry. The phospho-EGFR level was normal-ized to the total EGFR level (B) and the phospho-PDGFRb level was normalized with total PDGFRb(D). The levels of EGFR and PDGFRb were normal-ized with b-actin (C and E). Values are mean 6 S.D.(n = 6). Bars with different lowercase letters (a–d) aresignificantly different from one another (P , 0.01).

Fig. 5. SRT1720 enhances phosphorylation of EGFR and PDGFRb in cultured renal interstitial fibroblasts. NRK-49F cells were cultured in 2.5% fetalbovine serum containing medium and then incubated with different concentrations of SRT1720 (0–2 mM) for 36 hours (A–C). Cell lysates were preparedand subjected to immunoblot analysis with antibodies for phospho-EGFR (Tyr1068), EGFR, phospho-PDGFRb (Tyr751), PDGFRb, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (A). Representative immunoblots from three experiments are shown. The phospho-EGFR, phospho-PDGFRb,EGFR, PDGFRb, and GAPDHwere quantified by densitometry. (B) The phosphorylated EGFR and PDGFRbwere normalized to total protein levels. (C)The levels of EGFR and PDGFRb were normalized with GAPDH. Values are mean 6 S.D. of three independent experiments. Bars with differentlowercase letters (a and b) are significantly different from one another (P , 0.01).

Sirt1 Activation Aggravates Renal Fibrosis 147

at ASPE

T Journals on A

ugust 11, 2020jpet.aspetjournals.org

Dow

nloaded from

We further examined the effect of SRT1720 on the phos-phorylation and expression of STAT3, AKT, and ERK1/2in cultured renal fibroblasts. As shown in Fig. 7, SRT1720treatment at 1 and 2 mM remarkably increased the phos-phorylation level of STAT3 (Fig. 7, A and B). Similarly,SRT1720 at 2 mMalso enhanced AKT phosphorylation (Fig. 7,A and C); however, phosphorylation of ERK1/2 was notaffected by SRT1720 (Fig. 7, A and D). In addition, SRT1720treatment resulted in a decrease of total AKT (Fig. 7, A andC), but not total STAT3 (Fig. 7, A and B) or ERK1/2 (Fig. 7, Aand D), levels in cultured renal fibroblasts. Collectively, these

data indicate that SIRT1 activation by SRT1720 regulatesphosphorylation of STAT3 and AKT, but not ERK1/2, sug-gesting that SRIT1 has a diverse role in regulating activationof intracellular signaling pathways.

DiscussionSIRT-activating compounds such as resveratrol have been

widely studied for their ability to treat aging-related disordersin metabolic, cardiovascular, and neurodegenerative diseases(Stünkel and Campbell, 2011; Villalba and Alcain, 2012).

Fig. 6. SRT1720 enhances STAT3 phosphorylationin obstructed kidneys. Kidney tissue lysates wereprepared and subjected to immunoblot analysis withantibodies for phospho-STAT3 (Tyr705), STAT3,phospho-AKT (Ser473), AKT, phospho-ERK1/2,ERK1/2, or b-actin (A). The levels of phosphorylatedand total proteins were quantified by densitometry.The levels of phosphorylated proteins were normal-ized to their corresponding total protein (B, D, and F).The levels of STAT3, AKT, and ERK1/2 werenormalized with b-actin (C, E, and G). Values aremean 6 S.D. (n = 6). Bars with different lowercaseletters (a–d) are significantly different from oneanother (P , 0.01).

148 Ponnusamy et al.

at ASPE

T Journals on A

ugust 11, 2020jpet.aspetjournals.org

Dow

nloaded from

However, due to the poor specificity and bioavailability ofresveratrol, several molecules that are structurally unrelatedto resveratrol have been developed to stimulate SIRT activities,which are more potent and specific than resveratrol. SRT1720is a newly developed and effective SIRT1 activator that hasbeen shown to have the potential to treat diabetes and otherdiseases (Dong, 2012). In this study, we examined the pharma-cological effect of SRT1720 on the development of renal fibrosisand activation of renal fibroblasts in a mouse model of UUOand cultured renal interstitial fibroblasts. Our results showedthat activation of SIRT1 with SRT1720 potentiated thesepathologic processes. Moreover, exposure of cultured renalinterstitial fibroblasts to YK-3-237, another SIRT1 activator,also promoted their activation as demonstrated by increasedexpression of a-SMA and fibronectin. These results providestrong evidence that SIRT1 plays an essential role in medi-ating renal fibrogenesis after chronic kidney injury.Our findings are contrary to a previous report in which

activation of SIRT1 was demonstrated to be an antifibroticmechanism in the kidney by using resveratrol (Li et al., 2010).Currently, the molecular basis behind the striking incon-sistencies generated by these two SIRT1 activators in terms offibrosis regulation remains unclear. One possible explanationis that resveratrol acts on other targets rather than on SIRT1.In this regard, it has been reported that resveratrol has

a property enabling it to inhibit the activity of class I, II, andIV HDACs (Venturelli et al., 2013). Because blocking class I/IIHDACs with their specific inhibitors or siRNA have beenshown to suppress renal fibroblast activation (Pang et al.,2009, 2011; Liu et al., 2013) and because those class I/IIHDACs inhibitors are also effective in attenuating renalfibrosis (Pang et al., 2009; Liu et al., 2013), resveratrol-induced inhibition of these enzymes may override its role asthe activator of SIRT1, thereby exhibiting antifibrotic effects.Structural and chemical studies indicate that SRT1720 is100-fold more potent in stimulating SIRT1 activity whencompared with resveratrol (Villalba and Alcain, 2012).SRT1720-induced enhancement of the fibrotic process may

be involved in the activation EGFR and PDGFRb and theirdownstream signaling pathways. Previous studies from ourlaboratory and those from other groups have demonstratedthat sustained activation of EGFR and PDGFRb is associatedwith the activation of renal fibroblasts, expression ofprofibrotic factors, and uncontrolled accumulation of ECMin the interstitial space (Pang et al., 2010; LeBleu and Kalluri,2011; Liu et al., 2011, 2012, 2013). Class I/II HDACs havebeen suggested to mediate activation of several growth factorreceptor signaling pathways, including EGFR and STAT3activity in the kidney after chronic injury (Bruzzese et al.,2011; Chou et al., 2011; Liu et al., 2013). Similar to this,inhibition of SIRT1 reduced the phosphorylation of EGFR andPDGFRb in cultured renal fibroblasts and fibrotic kidneys(Ponnusamy et al., 2014). All these studies provide strongevidence that HDACs, including SIRT1, can function asa positive regulator of cellular membrane receptors associatedwith the fibrotic process. Thus, stimulation of SIRT1 bySRT1720 would result in the activation of these profibroticsignaling molecules. The mechanism by which SRT1720enhances the phosphorylation of receptor tyrosine kinasesremains unknown. One possibility is that SRT1720-inducedSIRT1 activation reduces the activity of tyrosine phosphatase.In supporting this hypothesis, SIRT1 is found to suppress theexpression of protein tyrosine phosphatase 1B (Sun et al.,2007; Gagarina et al., 2010), a member of the dephosphor-ylating protein family, which negatively regulates the activityof growth signaling molecules including EGFR and PDGFRb(Gu et al., 2003; Ponnusamy et al., 2013). Nevertheless, wecannot rule out the possibility that SRT1720 can also stimulaterenal fibrosis through stimulation of TGF-b signaling activa-tion because it has been reported that activation of SIRT1increased Smad reporter activity, enhanced transcription ofTGF-b target genes, and promoted release of collagen, whereasknockdown of SIRT1 inhibited TGF-b/SMAD signaling andreduced collagen release in skin fibroblasts (Zerr et al., 2014).Further experiments are needed to examine the role of SIRT1in the regulation of this signaling pathway in renal interstitialfibroblasts.Interestingly, SRT1720 administration increased the level

of phosphorylated and total STAT3 in both sham-operatedand fibrotic kidney. The structural analysis of STAT3revealed that both phosphorylation (Tyr705) and acetylation(Lys685) are located in the SH2 domain of the STAT3 protein.Therefore, acetylation and phosphorylation may work mutu-ally to regulate STAT3 activity (O’Shea et al., 2005). As such,SRT1720-mediated increase of SIRT1 activity may promotethe phosphorylation of STAT3 by inhibiting its acetylation inaddition to its influence on the protein tyrosine phosphatase

Fig. 7. SRT1720 enhances STAT3 phosphorylation in cultured renalinterstitial fibroblasts. NRK-49F cells were cultured in 2.5% fetal bovineserum containing medium and then with different concentrations ofSRT1720 (0–2 mM) for 36 hours (A–D). Cell lysates were prepared andsubjected to immunoblot analysis with antibodies for phospho-STAT3(Tyr705), STAT3, phospho-AKT (Ser473), AKT, phospho-ERK1/2, ERK1/2, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (A). Represen-tative immunoblots from three experiments are shown. The phosphorylatedand total proteins were quantified by densitometry and phosphorylatedprotein levels were normalized to their corresponding total protein level.The levels of total STAT3, AKT, and ERK1/2 were normalized with GAPDH(B–D). Values aremean6S.D. of three independent experiments. Barswithdifferent lowercase letters (a and b) are significantly different from oneanother (P , 0.01).

Sirt1 Activation Aggravates Renal Fibrosis 149

at ASPE

T Journals on A

ugust 11, 2020jpet.aspetjournals.org

Dow

nloaded from

system. On the other hand, SRT1720-mediated SIRT1 activa-tion may regulate STAT3 through suppressing the activity/expression of suppressor of cytokine signaling (SOCS) familymembers such as SOCS1 and SOCS3, the negative regulatorsof STAT3. In fact, these two SOCS not only promote dephos-phorylation but also ubiquitination and degradation of theSTAT family of proteins (Kile et al., 2002; Croker et al., 2008).Therefore, SIRT1-mediated inhibition of SOCS1/3 may abolishthe STAT3 degradation process, thereby increasing the stabil-ity of STAT3. As a result, both phosphorylated and total STAT3levels are increased. A further detailed study should be carriedout to unveil the mechanism of SRT1720-induced up-regulationof phosphorylated and total STAT3 levels.It is quite surprising that SRT1720 treatment reduced the

total AKT level in both fibrotic and sham-operated kidneyswithout affecting the phosphorylation of AKT (Ser473), whichresults in an increase in the ratio of pAKT to AKT. In general,phosphorylation of Ser473 at the hydrophobic motif site isnecessary for full activation of the AKT molecule, and it is alsothe major site engaged in many physiologic and pathologicfunctions of AKT (Liao and Hung, 2010). Recent studiesindicated that the AKT activity is controlled by the negativefeedback loop, which is triggered by hyperactivation of AKT,and this hyperactivation has the capacity to regulate AKTstability and/or expression (Hart and Vogt, 2011; Wu et al.,2011). In supporting this, Wu et al. (2011) found that consec-utive activation of AKT by Ser473 phosphorylation pro-motes its rapid degradation by a polyubiquitination-dependentproteosomal pathway, thereby turning off the AKT signaling(Wu et al., 2011). Given that SIRT1 promotes AKT phosphor-ylation through deacetylation (Horio, 2012; Li et al., 2013),SRT1720-induced SIRT1 activity might stimulate sustainedactivation of AKT, which in turn leads to degradation of AKTand reduction of total AKT levels.SIRT activity has been linked to metabolic control, cell sur-

vival, DNA repair, development, neuroprotection, and healthyaging (Stünkel and Campbell, 2011; Villalba and Alcain, 2012).Because SIRT activation could have beneficial effects on humandiseases, there is growing interest in the discovery of smallmolecules able to stimulate SIRT activity. Several smallmolecule activators of SIRT1 that are structurally unrelatedto resveratrol have been developed. Among them, SRT1720is one of the most effective SIRT1 activators. The role ofSIRT1720 in treating insulin resistance, diabetes, and otherdiseases has been tested in animal models, and drugs similarto SRT1720 are currently in human clinical trials (Villalba andAlcain, 2012). However, due to the fibrosis-promoting effect ofSIRT activators, large doses and long-term application shouldbe avoided when they are used clinically in the future.In summary, this is the first study to demonstrate that

activation of SIRT1 by SIRT1720 potentiates renal fibroblastactivation and renal fibrogenesis. The profibrotic effects ofSIRT1720 are associated with activation of EGFR andPDGFRb and their downstream signaling molecules STAT3and AKT. On this basis, caution should be used in theapplication of SIRT1 activators in order to avoid induction ofkidney fibrotic disorders or acceleration of pre-existingchronic kidney disease when they are used for renal diseases.

Authorship Contributions

Participated in research design: Ponnusamy, Zhao, S. Zhuang.Conducted experiments: Ponnusamy, Zhou, Tolbert.

Contributed new reagents or analytic tools: Ponnusamy, Tolbert.Performed data analysis: Ponnusamy, Zhou.Contributed to the writing of the manuscript: Ponnusamy,

M. A. Zhuang, Bayliss, S. Zhuang.

References

Bonner JC (2004) Regulation of PDGF and its receptors in fibrotic diseases. CytokineGrowth Factor Rev 15:255–273.

Bruzzese F, Leone A, Rocco M, Carbone C, Piro G, Caraglia M, Di Gennaro E,and Budillon A (2011) HDAC inhibitor vorinostat enhances the antitumor effect ofgefitinib in squamous cell carcinoma of head and neck by modulating ErbB re-ceptor expression and reverting EMT. J Cell Physiol 226:2378–2390.

Chou CW, Wu MS, Huang WC, and Chen CC (2011) HDAC inhibition decreases theexpression of EGFR in colorectal cancer cells. PLoS ONE 6:e18087.

Croker BA, Kiu H, and Nicholson SE (2008) SOCS regulation of the JAK/STATsignalling pathway. Semin Cell Dev Biol 19:414–422.

Di Pascoli M, Diví M, Rodríguez-Vilarrupla A, Rosado E, Gracia-Sancho J, VilasecaM, Bosch J, and García-Pagán JC (2013) Resveratrol improves intrahepatic en-dothelial dysfunction and reduces hepatic fibrosis and portal pressure in cirrhoticrats. J Hepatol 58:904–910.

Dong XC (2012) Sirtuin biology and relevance to diabetes treatment. Diabetes Manag(Lond) 2:243–257.

Dong YJ, Liu N, Xiao Z, Sun T, Wu SH, Sun WX, Xu ZG, and Yuan H (2014) Renalprotective effect of sirtuin 1. J Diabetes Res 2014:843786.

Fan H, Yang HC, You L, Wang YY, He WJ, and Hao CM (2013) The histone deace-tylase, SIRT1, contributes to the resistance of young mice to ischemia/reperfusion-induced acute kidney injury. Kidney Int 83:404–413.

Gagarina V, Gabay O, Dvir-Ginzberg M, Lee EJ, Brady JK, Quon MJ, and Hall DJ(2010) SirT1 enhances survival of human osteoarthritic chondrocytes by repressingprotein tyrosine phosphatase 1B and activating the insulin-like growth factor re-ceptor pathway. Arthritis Rheum 62:1383–1392.

Gu F, Dubé N, Kim JW, Cheng A, Ibarra-Sanchez MdeJ, Tremblay ML, and BoisclairYR (2003) Protein tyrosine phosphatase 1B attenuates growth hormone-mediatedJAK2-STAT signaling. Mol Cell Biol 23:3753–3762.

Hart JR and Vogt PK (2011) Phosphorylation of AKT: a mutational analysis. Onco-target 2:467–476.

Horio Y (2012) Diabetes: insulin signal meets SIRT1 at AKT. Nat Rev Endocrinol 8:131–132.

Imanishi S, Hayashi R, Ichikawa T, Suzuki K, Sasahara M, Kondo T, Ogawa H,and Tobe K (2012) SRT1720, a SIRT1 activator, aggravates bleomycin-inducedlung injury in mice. Food Nutrition Sci 3:157–163.

Kile BT, Schulman BA, Alexander WS, Nicola NA, Martin HM, and Hilton DJ (2002)The SOCS box: a tale of destruction and degradation. Trends Biochem Sci 27:235–241.

Kitada M, Kume S, Takeda-Watanabe A, Kanasaki K, and Koya D (2013) Sirtuinsand renal diseases: relationship with aging and diabetic nephropathy. Clin Sci(Lond) 124:153–164.

Lan A and Du J (2015) Potential role of Akt signaling in chronic kidney disease.Nephrol Dial Transplant 30:385–394.

LeBleu VS and Kalluri R (2011) Blockade of PDGF receptor signaling reducesmyofibroblast number and attenuates renal fibrosis. Kidney Int 80:1119–1121.

Li J, Qu X, Ricardo SD, Bertram JF, and Nikolic-Paterson DJ (2010) Resveratrolinhibits renal fibrosis in the obstructed kidney: potential role in deacetylation ofSmad3. Am J Pathol 177:1065–1071.

Li XH, Chen C, Tu Y, Sun HT, Zhao ML, Cheng SX, Qu Y, and Zhang S (2013) Sirt1promotes axonogenesis by deacetylation of Akt and inactivation of GSK3. MolNeurobiol 48:490–499.

Liao Y and Hung MC (2010) Physiological regulation of Akt activity and stability. AmJ Transl Res 2:19–42.

Liu N, Guo JK, Pang M, Tolbert E, Ponnusamy M, Gong R, Bayliss G, Dworkin LD,Yan H, and Zhuang S (2012) Genetic or pharmacologic blockade of EGFR inhibitsrenal fibrosis. J Am Soc Nephrol 23:854–867.

Liu N, He S, Ma L, Ponnusamy M, Tang J, Tolbert E, Bayliss G, Zhao TC, Yan H,and Zhuang S (2013) Blocking the class I histone deacetylase ameliorates renalfibrosis and inhibits renal fibroblast activation via modulating TGF-beta andEGFR signaling. PLoS ONE 8:e54001.

Liu N, Tolbert E, Pang M, Ponnusamy M, Yan H, and Zhuang S (2011) Suramininhibits renal fibrosis in chronic kidney disease. J Am Soc Nephrol 22:1064–1075.

Ludewig D, Kosmehl H, Sommer M, Böhmer FD, and Stein G (2000) PDGF receptorkinase blocker AG1295 attenuates interstitial fibrosis in rat kidney after unilateralobstruction. Cell Tissue Res 299:97–103.

Meran S and Steadman R (2011) Fibroblasts and myofibroblasts in renal fibrosis. IntJ Exp Pathol 92:158–167.

O’Shea JJ, Kanno Y, Chen X, and Levy DE (2005) Cell signaling. Stat acetylation—a key facet of cytokine signaling? Science 307:217–218.

Pacholec M, Bleasdale JE, Chrunyk B, Cunningham D, Flynn D, Garofalo RS,Griffith D, Griffor M, Loulakis P, Pabst B, et al. (2010) SRT1720, SRT2183,SRT1460, and resveratrol are not direct activators of SIRT1. J Biol Chem 285:8340–8351.

Pang M, Kothapally J, Mao H, Tolbert E, Ponnusamy M, Chin YE, and Zhuang S(2009) Inhibition of histone deacetylase activity attenuates renal fibroblast acti-vation and interstitial fibrosis in obstructive nephropathy. Am J Physiol RenalPhysiol 297:F996–F1005.

Pang M, Ma L, Gong R, Tolbert E, Mao H, Ponnusamy M, Chin YE, Yan H, DworkinLD, and Zhuang S (2010) A novel STAT3 inhibitor, S3I-201, attenuates renal in-terstitial fibroblast activation and interstitial fibrosis in obstructive nephropathy.Kidney Int 78:257–268.

150 Ponnusamy et al.

at ASPE

T Journals on A

ugust 11, 2020jpet.aspetjournals.org

Dow

nloaded from

Pang M, Ma L, Liu N, Ponnusamy M, Zhao TC, Yan H, and Zhuang S (2011) Histonedeacetylase 1/2 mediates proliferation of renal interstitial fibroblasts and expres-sion of cell cycle proteins. J Cell Biochem 112:2138–2148.

Pang M and Zhuang S (2010) Histone deacetylase: a potential therapeutic target forfibrotic disorders. J Pharmacol Exp Ther 335:266–272.

Ponnusamy M, Ma L, and Zhuang S (2013) Necrotic renal epithelial cell inhibitsrenal interstitial fibroblast activation: role of protein tyrosine phosphatase 1B. AmJ Physiol Renal Physiol 304:F698–F709.

Ponnusamy M, Zhou X, Yan Y, Tang J, Tolbert E, Zhao TC, Gong R, and Zhuang S(2014) Blocking sirtuin 1 and 2 inhibits renal interstitial fibroblast activation andattenuates renal interstitial fibrosis in obstructive nephropathy. J Pharmacol ExpTher 350:243–256.

Qin L and Han YP (2010) Epigenetic repression of matrix metalloproteinases inmyofibroblastic hepatic stellate cells through histone deacetylases 4: implication intissue fibrosis. Am J Pathol 177:1915–1928.

Rombouts K, Niki T, Greenwel P, Vandermonde A, Wielant A, Hellemans K, DeBleser P, Yoshida M, Schuppan D, Rojkind M, et al. (2002) Trichostatin A, a his-tone deacetylase inhibitor, suppresses collagen synthesis and prevents TGF-beta(1)-induced fibrogenesis in skin fibroblasts. Exp Cell Res 278:184–197.

Stünkel W and Campbell RM (2011) Sirtuin 1 (SIRT1): the misunderstood HDAC. JBiomol Screen 16:1153–1169.

Sun C, Zhang F, Ge X, Yan T, Chen X, Shi X, and Zhai Q (2007) SIRT1 improvesinsulin sensitivity under insulin-resistant conditions by repressing PTP1B. CellMetab 6:307–319.

Tampe B and Zeisberg M (2014) Contribution of genetics and epigenetics toprogression of kidney fibrosis. Nephrol Dial Transplant 29 (Suppl 4):iv72–iv79.

Tang J, Liu N, Tolbert E, Ponnusamy M, Ma L, Gong R, Bayliss G, Yan H,and Zhuang S (2013) Sustained activation of EGFR triggers renal fibrogenesisafter acute kidney injury. Am J Pathol 183:160–172.

Terzi F, Burtin M, Hekmati M, Federici P, Grimber G, Briand P, and Friedlander G(2000) Targeted expression of a dominant-negative EGF-R in the kidney reducestubulo-interstitial lesions after renal injury. J Clin Invest 106:225–234.

Van Beneden K, Mannaerts I, Pauwels M, Van den Branden C, and van Grunsven LA(2013) HDAC inhibitors in experimental liver and kidney fibrosis. FibrogenesisTissue Repair 6:1.

Venturelli S, Berger A, Böcker A, Busch C, Weiland T, Noor S, Leischner C,Schleicher S, Mayer M, Weiss TS, et al. (2013) Resveratrol as a pan-HDAC in-hibitor alters the acetylation status of histone [corrected] proteins in human-derived hepatoblastoma cells. PLoS ONE 8:e73097.

Villalba JM and Alcaín FJ (2012) Sirtuin activators and inhibitors. Biofactors 38:349–359.

Wu YT, Ouyang W, Lazorchak AS, Liu D, Shen HM, and Su B (2011) mTOR complex2 targets Akt for proteasomal degradation via phosphorylation at the hydrophobicmotif. J Biol Chem 286:14190–14198.

Yi YW, Kang HJ, Kim HJ, Kong Y, Brown ML, and Bae I (2013) Targeting mutantp53 by a SIRT1 activator YK-3-237 inhibits the proliferation of triple-negativebreast cancer cells. Oncotarget 4:984–994.

Zeisberg M and Neilson EG (2010) Mechanisms of tubulointerstitial fibrosis. J AmSoc Nephrol 21:1819–1834.

Zerr P, Palumbo-Zerr K, Huang J, Tomcik M, Sumova B, Distler O, Schett G,and Distler JH (2014) Sirt1 regulates canonical TGF-b signalling to control fibro-blast activation and tissue fibrosis. Ann Rheum Dis DOI:10.1136/annrheumdis-2014-205740 [published ahead of print].

Address correspondence to: Dr. Shougang Zhuang, Department of Nephrology,Shanghai East Hospital, Tongji University School of Medicine, 150 Jiamo Rd.,Shanghai 200120, China. E-mail: szhuang@lifespan.org

Sirt1 Activation Aggravates Renal Fibrosis 151

at ASPE

T Journals on A

ugust 11, 2020jpet.aspetjournals.org

Dow

nloaded from