apelin inhibits the development of diabetic nephropathy by regulating histone acetylation in akita...

J Physiol 592.3 (2014) pp 505–521 505

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Apelin inhibits the development of diabetic nephropathyby regulating histone acetylation in Akita mouse

Hong Chen1, Jianshuang Li2, Lihua Jiao1, Robert B. Petersen3, Jiong Li2, Anlin Peng4, Ling Zheng2

and Kun Huang1,5

1Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan 430030, China2College of Life Sciences, Wuhan University, Wuhan 430072, China3Departments of Pathology, Neuroscience and Neurology, Case Western Reserve University, Cleveland, OH 44106, USA4The Third Hospital of Wuhan, Wuhan 430060, China5Centre for Biomedicine Research, Wuhan Institute of Biotechnology, Wuhan 430074, China

Key points

� Diabetic nephropathy (DN) is a major complication of diabetes, key features of whichinclude glomerular mesangial expansion, hypertrophy, renal inflammation and accumulationof extracellular matrix proteins in the kidney.

� Histone acetylation plays an important role in the regulation of inflammation in DN.� We found that apelin-13, the most active member of the adipokine apelin group, decreased

glomerular filtration rate, proteinuria, glomerular hypertrophy and mesangial expansion,down-regulated histone acetylation and suppressed renal inflammation in Akita mouse, aspontaneous DN mouse model.

� In mesangial cell lines, apelin-13 treatment not only inhibited high glucose-induced histonehyperacetylation and inflammation factors, but also up-regulated histone deacetylase 1.

� These results revealed the possible mechanisms underlying the regulation of histone acetylationin DN and provide novel approaches to explore the beneficial effects of apelin-13 on DN.

Abstract Diabetic nephropathy is the primary cause of end-stage renal disease. Increasingnumbers of patients are suffering from this disease and therefore novel medications andtherapeutic approaches are urgently needed. Here, we investigated whether apelin-13, the mostactive member of the adipokine apelin group, could effectively suppress the development ofnephropathy in Akita mouse, a spontaneous type 1 diabetic model. Apelin-13 treatment decreaseddiabetes-induced glomerular filtration rate, proteinuria, glomerular hypertrophy, mesangialexpansion and renal inflammation. The inflammatory factors, activation of NF-κB, histoneacetylation and the enzymes involved in histone acetylation were further examined in diabetickidneys and high glucose- or sodium butyrate-treated mesangial cells in the presence or absenceof apelin-13. Apelin-13 treatment inhibited diabetes-, high glucose- and NaB-induced elevationof inflammatory factors, and histone hyperacetylation by upregulation of histone deacetylase 1.Furthermore, overexpression of apelin in mesangial cells induced histone deacetylation underhigh glucose condition. Thus, apelin-13 may be a novel therapeutic candidate for treatment ofdiabetic nephropathy via regulation of histone acetylation.

C© 2013 The Authors. The Journal of Physiology C© 2013 The Physiological Society DOI: 10.1113/jphysiol.2013.266411

506 H. Chen and others J Physiol 592.3

(Resubmitted 6 October 2013; accepted after revision 11 November 2013; first published online 18 November 2013)Corresponding authors L. Zheng: College of Life Sciences, Wuhan University, Wuhan 430072, China.Email: [email protected]; Kun Huang: Tongji School of Pharmacy, Huazhong University of Science andTechnology, Wuhan 430030, China. Email: [email protected]

Abbreviations A/G, albumin/globulin; AGEs, advanced glycation end-products; AMPK, AMP-activated protein kinase;DN, diabetic nephropathy; ER, endoplasmic reticulum; ESRD, end-stage renal disease; GFR, glomerular filtration rate;HAT, histone acetyltransferase; HDAC, histone deacetylase; HG, high glucose; ICAM-1, intercellular adhesion molecule1; KI, kidney index; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemotactic protein 1; NaB, sodiumbutyrate; NG, normal glucose; PKC, protein kinase C; TGF-β, transforming growth factor-β; 2K1C, two-kidney-one-clip;VCAM-1, vascular cell adhesion molecule 1..

Introduction

Diabetic nephropathy (DN), a common microvascularcomplication of diabetes, is characterized by glomerular,tubular and tubulointerstitial injury resulting fromhyperglycaemic conditions (Cooper, 2001). The earliestpathological features of DN include glomerular hyper-trophy and thickening of the glomerular basementmembrane (Cooper, 2001). As the disease progresses,glomerular hyperfiltration leads to albuminuria, andeventually to end-stage renal failure (Susztak & Bottinger,2006). With the dramatic increase in the number ofdiabetic patients globally over the past two decades, DN isnow the primary cause of end-stage renal disease (ERSD)and a major cause of morbidity and mortality in diabeticpatients (Susztak & Bottinger, 2006). Currently, dialysisand renal replacement therapy are the only treatmentsfor the late stage of DN. However, the cost of thesetreatments is extremely high and thus there is a great needfor the development of novel medications and therapeuticapproaches.

Several mechanisms have been implicated in thepathogenesis of DN, including increased oxidative stress,elevated levels of transforming growth factor-β (TGF-β),protein kinase C (PKC) and mitogen-activated proteinkinases (MAPKs), as well as the activation of thereceptor for advanced glycation end-products (RAGE) andinflammatory transcription factors like NF-κB (Schena& Gesualdo, 2005). Recently, chronic inflammation hasemerged as a key etiology of DN (Navarro-Gonzalez& Mora-Fernandez, 2008). Inflammatory chemokineMCP-1 (monocyte chemotactic protein 1), inflammatoryenzyme iNOS (inducible nitric oxide synthases) andadhesion molecules such as ICAM-1 (intercellularadhesion molecule 1) and VCAM-1 (vascular cell adhesionmolecule 1), are thought to play important roles in thedevelopment of DN (Chow et al. 2005, 2007; Tarabraet al. 2009; Alkhalaf et al. 2012). Therefore, understandinghow inflammation is mediated in the development of DNshould identify novel therapeutic targets.

Increasing evidence suggests that histone acetylationplays an important role in the regulation of inflammation

(Zager et al. 2011; Shanmugam & Sethi, 2012). Increasedhistone acetylation, especially increased levels of ac-H3K9and ac-H3K14, are usually associated with gene activation,including the inflammatory cytokines and chemokines(Sayyed et al. 2010; Jin et al. 2011). Inhibition ofhistone acetylation by minocycline, garcinol or siRNA ofp300 inhibits the high glucose-induced inflammation incultured retinal cells (Kadiyala et al. 2012; Wang et al.2012). However, the exact role of histone acetylation inthe development of DN remains unknown.

The apelin family of adipokines is derived from a77-residue preproprotein with lengths varying from 12to 36 residues (Tatemoto et al. 1998; Chen et al.2003). Apelin-13, the most active member of the apelingroup, performs multiple biological functions, includingregulation of glucose balance, blood pressure and foodintake (Reaux et al. 2001, 2002; Dray et al. 2008). Acuteadministration of apelin-13 improves glucose tolerance,and increases glucose utilization in normal and type 2diabetic mice via activation of AMPK (AMP-activatedprotein kinase) and AKT (also known as protein kinase B,PKB) signalling pathways (Dray et al. 2008). In additionto its beneficial effects on type 2 diabetes, apelin-13 alsoimproves the morphology of pancreatic islets by reducingdiabetes-induced ER (endoplasmic reticulum) stress inthe pancreas of Akita mice in C57BL/6 background (Chenet al. 2011), which is a well-characterized mouse modelof type 1 diabetes with spontaneous nephropathy (Proctoret al. 2006; Yi et al. 2012). That apelin has a beneficial effectof on renal disease is also suggested by several studies. Forexample, apelin has been found to lower blood pressure inthe two-kidney-one-clip (2K1C) hypertension rat modelthrough its effect on vascular relaxation (Soltani Hekmatet al. 2011). Apelin has also been observed to protectcells at a histopathological level in a rat model of renalischaemia and reperfusion injury (Sagiroglu et al. 2012).Furthermore, subcutaneous injection of apelin-13 reducesglomerular hypertrophy and renal inflammation in Ove26mice, a model of type 1 diabetes (Day et al. 2013).However, the mechanisms by which apelin-13 protectsagainst renal diseases are still unclear.

C© 2013 The Authors. The Journal of Physiology C© 2013 The Physiological Society

J Physiol 592.3 Apelin inhibits the development of diabetic nephropathy 507

To explore the mechanisms underlying the beneficialeffects of apelin-13 on DN, in the present study, apelin-13was used to treat Akita mice for 2 months. In aparallel study, mesangial cell lines were cultured underdiabetic-like conditions and also treated with apelin-13.We sought to discover: (1) whether the apelin-13 hassimilar protective effects on the development of DN inAkita mouse; (2) whether apelin-13 inhibits diabetes-or high glucose- induced inflammation by regulationof histone acetylation in vivo and in vitro, and whichhistone acetylation regulatory enzyme is responsiblefor the effects of apelin-13 on histone acetylation;and (3) whether elevation of histone acetylation incultured mesangial cells promotes inflammation, andwhether apelin-13 has similar inhibitory effects on histonehyperacetylation-induced inflammation.

Methods

Ethical approval

Mice were handled according to the Guidelines of theChina Animal Welfare Legislation, as approved by theCommittee on Ethics in the Care and Use of LaboratoryAnimals of College of Life Sciences, Wuhan University.

Animals

Breeding pairs of Akita (Insulin2+/−) mice on a C57BL/6background were obtained from the Model AnimalResearch Center of Nanjing University, as previouslyreported (Chen et al. 2011). Animals were housed inventilated microisolator cages with free access to waterand food in a temperature-controlled room (22 ± 2°C)with a 12 h light–dark cycle. Male Akita mice (Insulin2+/−)and their non-diabetic littermates (Insulin2+/+, wild type(WT)) were used in the present study. The experimentaldesign is shown in Fig. 1A. Forty-two mice wereseparated into four groups: PBS-treated WT mice (WT);apelin-treated WT mice (WT+Ap); PBS-treated Akitamice (AK); and apelin-treated Akita mice (AK+Ap). Theapelin-13 peptide was chemically synthesized, purified andcharacterized as previously described (Chen et al. 2011).The experiments were started at 9 weeks of age, since maleAkita mice begin to show hyperglycaemia at 4–7 weeks ofage (Yoshioka et al. 1997; Toque et al. 2013). Therefore,the symptoms of animals at the start of experiments aresimilar to those of early-stage diabetic patients. Apelin-13was administrated at a dose of 400 pmol (kg body weight)–1

to Akita mice via tail vein injection twice per day. Apelinat this dosage had been shown to mildly reduce fastingblood glucose levels in Akita mice after injection for7 days in our pilot studies (data not shown). In the pre-sent study, apelin-13 or PBS was injected via the tail veinfrom the most distal end to the root of the tail as pre-

viously described (Takeuchi et al. 1984; Chen et al. 2011).Animals underwent terminal anaesthesia at 19 weeks withchloral hydrate (IP, 500 mg kg−1) to enable harvesting ofkidneys and serum, which resulted in subsequent death byexsanguinations.

Measurement of biochemical parameters

Twenty-four hour urine samples were collected inmetabolic cages 1 day before killing, and the volume ofcollected urine was measured. Serum levels of creatinine,albumin and globin were analysed with a Siemens ADVIA2400 automatic biochemistry analyser using a creatininereagent kit, an albumin reagent kit, or a globin reagentkit (all from Fuxing Changzheng Medical Inc., Shanghai,China). The urine levels of creatinine and total proteinwere measured with an Olympus AU2700 automaticbiochemistry analyser using a creatinine reagent kit(Fuxing Changzheng Medical Inc.) or a total proteinreagent kit (Great Wall Clinical Reagent Inc., Baoding,China).

Cell culture

The rat kidney mesangial cell line, HBZY-1 (obtained fromChina Center for Type Culture Collection, Wuhan, China),and the mouse mesangial cell line, MES13 (obtained fromShanghai Institute of Cell Resource Center, Shanghai,China) were cultured in normal glucose (NG)–DMEMmedia containing 5.5 mM glucose (Hyclone, Palo Alto,CA, USA) with 10% FBS in a 37°C incubator with 5%CO2. At 80% cell confluence, the media was replacedwith either high glucose (HG)–DMEM media (Hyclone)containing 25 mM glucose with or without different dosesof apelin-13 (30 pM and 300 pM), or NG–DMEM media.After being cultured under different conditions for onemore day, cells were harvested and analysed. In anotherset of experiments, HBZY-1 cells were treated with 5 mM

sodium butyrate (NaB, an inhibitor of HDACs) underthe NG conditions with or without different dosages ofapelin-13 and the cells were collected 24 h after treatment.

Immunofluorescence staining

After fixation and blocking with 2% BSA, a primary anti-body against HDAC1 (1:500 dilution, Abcam, Cambridge,MA) was applied to the cells for 4°C overnight. Afterwashing with PBS three times, the cells were incubatedwith the appropriate secondary antibody. Cells werecovered with 4′,6-diamidino-2-phenylindole (DAPI) dyeand anti-fading medium after extensive washing withPBS, and imaged with an Olympus BX60 Microscope(Olympus, Japan).



Transfection

HBZY-1 cells were plated into 6-well plates and trans-fected the next day with either pPCAGSIH–β-gal orpPCAGSIH–apelin plasmids (kind gifts from Dr Takakura,Osaka University, Japan) using Fugene HD transfectionreagent (Promega, Madison, WI, USA) according to themanufacturer’s instruction.

Renal histology

The kidneys were rapidly dissected and fixed in 10%buffered formalin at 4°C overnight. The kidneys wereembedded in paraffin and were sectioned at 5 μmthickness on positively charged slides. Sections werestained with periodic acid–Schiff and Haematoxylin(PASH). The renal histology was examined in a blindedmanner. High resolution pictures of 35–40 glomeruliper animal were taken using an Olympus BX60 micro-scope equipped with a digital CCD. The glomerularcross-sectional areas (Ag) were measured using ImagePlus6.0 software (Media Cybernetics, Bethesda, Maryland).The glomerular volume (Vg) was calculated usingWeibel-Gomez formula (Lane et al. 1992), and was furthernormalized to the mean volume (set as one) of the wildtype group.

Immunohistochemical studies

Paraffin-embedded sections were deparaffinized andrehydrated as previously reported (Chen et al. 2011; Dinget al. 2013). Sections were incubated with 3% H2O2 for5 min to quench endogenous peroxidase activity. Afterblocking with 2% goat serum in PBST, primary antibodiesincluding type IV collagen (1:200 dilution, Rockland,Gilbertsville, PA, USA), apelin (1:200 dilution, Abcam),and F4/80 (1:200 dilution, Santa Cruz, Biotechnology,Inc., Dallas, TX, USA) were applied to the sections over-night at 4°C. After washing with PBST, sections wereincubated with biotinylated anti-rabbit or anti-rat anti-bodies (Vector laboratories, Burlingame, CA, USA) for1 h at room temperature. Positive staining was visualizedusing DAB substrate (Vector laboratories) following theABC kit protocol (Vector laboratories). Pictures of at least35 different glomeruli from each sample were taken usingan Olympus BX60 microscope with a digital CCD. Thedata are presented as fold changes compared to the levelof wild type, which is set as one-fold.

qRT-PCR

RNA was extracted from cultured cells using RNAisoPlus (Takara Biotechnology Co., Dalian, China) as pre-viously reported (Wang et al. 2012). cDNA synthesis was

performed using the M-MLV First Stand Kit (Invitrogen,Carlsbad, CA, USA). Primer sequences of target genesare provided in Supplemental Table S1. Real time PCRwas performed using a CFX96 Touch Real-Time PCRDetection System (Bio-Rad, Hercules, CA, USA). Theformation of a single product from each primer set wasconfirmed by the observation of only one peak in themelting curve for each reaction. 18S rRNA was used as aninternal control. The relative difference was expressed asthe fold change calculated by the 2−��CT method.

Western blots

Freshly isolated kidney cortex or cultured cells weresonicated in ice-cold RIPA buffer (Beyotime, China) andprotein concentrations were quantified as described (Liet al. 2012a; Wang et al. 2012). Then 20–80 μg ofproteins from each sample were separated by SDS–PAGE.The proteins were transferred onto PVDF membranesfor immunodetection. The list of antibodies used inthe present study is provided in Supplemental Table S2.The expression levels of target proteins were quantifiedusing Quantity One 1-D Analysis Software (Bio-Rad).The protein expression levels were quantified relative toβ-actin in the same sample and were further normalizedto the respective control group, which was set at one. Toensure the reproducibility of the Western blots, all samplesets were tested with Western blot for three batches. In thequantitative figures, the mean densities of three individualmeasurements were used, and the error bars stand for thedeviations of the three experimental data sets.

Chromatin immunoprecipitation (ChIP) assay

The kidney cortices were cross-linked using 1%formaldehyde and stopped by adding glycine, and theChIP assay was performed as previously described (Liet al. 2012b). Chromatin was immunoprecipitated withanti-ac-K antibody (Cell Signaling Technology, Inc.,Danvers, MA, USA). The purified DNA was detectedby standard PCR. Primer sequences are provided inSupplemental Table S1. The input samples were used asthe internal control for comparison between samples.

Statistical analysis

The data were expressed as means ± SEM. Statisticalsignificance was determined by analysing the data withthe non-parametric Kruskal–Wallis test, followed bythe Mann–Whitney test. Differences were consideredstatistically significant at a P value < 0.05.



Results

Administration of apelin-13 inhibits diabetes-inducedrenal dysfunction and renal histological changes

The results of kidney weight, kidney index (KI),proteinuria, albumin/globulin (A/G) and glomerularfiltration rate (GFR) for different experimental groupsare summarized in Table 1. Compared to WT mice, Akitamice showed significant increases in kidney weight, KI,proteinuria, GFR, and a significant decrease in the A/Gratio. For WT mice, apelin-13 treatment showed no effectson kidney weight, KI and the A/G ratio; however, it ledto a significant reduction on the levels of proteinuriaand GFR; but for Akita mice, apelin-13 treatment notonly suppressed diabetes-induced increases in kidneyweight, KI, proteinuria and GFR, but also normalized thediabetes-induced decrease in the ratio of A/G.

An immunohistochemsitry study was performed toinvestigate the apelin level in the kidneys of differentexperimental groups. The number of apelin-positivestained cells was dramatically reduced in the glomeruliand convoluted tubules of Akita mice (Fig. 1B and

C). Apelin-13 treatment significantly inhibited thediabetes-induced decrease of apelin level in the kidneysof Akita mice, but the treatment showed no obvious effecton apelin level in the kidneys of WT mice (Fig. 1B andC). However, the Western blot analysis of angiotensinreceptor-related G protein-coupled receptor (APJ), thereceptor of apelin-13, suggested the protein levels ofAPJ were unchanged among all experimental groups(Supplemental Fig. S1).

Typical glomerular damage was observed in thekidneys of Akita mice, including glomerular hyper-trophy and mesangial matrix accumulation (Fig. 2A).These diabetes-induced morphological changes weresignificantly reduced by apelin-13 treatment (Fig. 2A).Quantitative analysis suggested that the glomerular areawas significantly larger in Akita mice than in WTmice (1.5-fold increase; Fig. 2B). Apelin-13 significantlyreduced diabetes-induced glomerular hypertrophy (27%decrease compared to Akita mice; Fig. 2B). Moreover,Akita mice showed a 3.4-fold increase in type IVcollagen staining in the glomeruli compared to WTmice (Fig. 2C and D). Akita mice treated with apelin-13

Figure 1. Experimental design and apelin immunohistochemistry stainingA, experimental design. Representative pictures of apelin staining (B) and quantitative analysis of apelin-stainedarea in the glomeruli (C) of different experimental groups. WT, wild-type mice, n = 9; WT + Ap, apelin-13-treatedwild-type mice, n = 10; AK, Akita mice, n = 9; AK + Ap, apelin-13-treated Akita mice, n = 11. Bar = 50 μm.Brown colour indicates apelin positive staining. ∗P < 0.05 compared to WT mice; #P < 0.05 compared to Akitamice.



Table 1. Effects of apelin-13 treatment

WT (n) WT + Ap (n) AK (n) AK + Ap (n)

Kidney weight (mg) 145.5 ± 15.5 (12) 131.5 ± 6.7 (10) 202.2 ± 16.3∗ (9) 146.0 ± 12.4# (11)KI (mg g−1, %) 5.6 ± 0.5 (12) 6.2 ± 0.2 (10) 9.0 ± 0.9∗ (9) 6.8 ± 0.7# (11)A/G (serum) 1.8 ± 0.2 (12) 1.7 ± 0.1 (10) 1.3 ± 0.5∗ (9) 1.8 ± 0.1# (8)Urine volume (ml) 0.4 ± 0.1 (6) 0.4 ± 0.1 (3) 17.4 ± 4.5∗ (9) 6.5 ± 3.9# (11)Proteinuria (mg (24 h)–1) 3.2 ± 1.0 (6) 1.0 ± 0.6∗ (3) 14.3 ± 7.9∗ (9) 8.9 ± 7.1# (11)GFR (Ccr, ml min−1) 0.06 ± 0.02 (6) 0.02 ± 0.01∗ (3) 1.8 ± 1.6∗ (9) 0.2 ± 0.1# (8)

WT, wild-type mice; WT + Ap, apelin-13-treated wild-type mice; AK, Akita mice; AK + Ap, apelin-13-treated Akita mice. KI: kidneyweight (mg)/body weight (g), A/G: albumin/globulin; GFR: glomerular filtration rate; ∗P < 0.05 compared to WT mice; #P < 0.05compared to Akita mice. Due to limited amount of urine collected from the WT group and WT + apelin group, only 6 and 3 animals,respectively, from each group gave enough volume of urine samples that warrant further measurements, therefore the n numbers ofurine volume, proteinuria and GFR of these two groups were 6 and 3, respectively.

showed a significant reduction of the diabetes-inducedaccumulation of type IV collagen in the glomeruli (52%decrease compared to Akita mice; Fig. 2C and D). However,administration of apelin-13 on the WT mice showedno effect on either glomerular area or type IV collagenstaining in the kidneys.

Apelin-13 treatment inhibits diabetes-inducedhistone hyperacetylation in the kidney byupregulating HDAC1

Histone acetylation is known to activate gene transcriptionby promoting the access of transcription factors totheir DNA target sites. The levels of total lysine acetyl-ation (ac-K), ac-H3K9, ac-H3K18 and ac-H3K23 wereall significantly increased in the renal cortex of Akitamice compared to WT mice (2.1-fold, 3.3-fold, 9.7-foldand 2.5-fold increase, respectively; Fig. 3A and B). Inapelin-13-treated Akita mice, diabetes-induced elevationof histone acetylation levels was reduced to near baselinelevels (Fig. 3A and B). However, apelin-13 treatment onWT mice showed no significant effect on either ac-K orspecific acetylated histone H3 sites, such as K9 and K18(Supplemental Fig. S2A and B).

Since total histone acetylation levels are tightlycontrolled by the balance of histone acetyltransferases(HATs) and histone deacetylases (HDACs), the levelsof two HDACs (HDAC1 and HDAC2) and two HATs(PCAF and GCN5) were determined. Compared toWT mice, HDAC1 was significantly decreased (55%reduction; Fig. 3C and D), while PCAF was sharplyincreased (25.9-fold increase; Fig. 3E and F) in the renalcortex of Akita mice; whereas the expression levels ofHDAC2 and GCN5 were similar between Akita miceand WT mice (Fig. 3C–F). Only the expression level ofHDAC1 was affected by apelin-13 treatment in Akita mice(3.5-fold increase compared to Akita mice), which was stillsignificantly higher than that of the WT group (Fig. 3D).However, apelin-13 treatment of normal mice showed nosignificant effect on expression levels of either HDCA1/2or PCAF/GCN5 (Supplemental Fig. S2C and D).

Apelin-13 treatment inhibits diabetes-induced renalinflammation in Akita mice

The levels of several inflammatory molecules wereexamined. Compared to WT mice, Akita mice showedsignificantly elevated levels of MCP-1, ICAM-1 andiNOS (7.8-fold, 2.3-fold, 4.0-fold increase, respectively;Fig. 4A and B), suggesting a significant increase inrenal inflammation. Apelin-13 significantly reduceddiabetes-induced increases in MCP-1, ICAM-1 and iNOSlevels of Akita mice (Fig. 4A and B). To further examine theinhibitory effect of apelin-13 on inflammation, F4/80 (amacrophage marker) staining was performed. Comparedto WT mice, Akita mice showed a significant accumulationof macrophages, while apelin-13 dramatically reduceddiabetes-induced elevation of macrophage infiltration inAkita mice (Fig. 4C and D).

ChIP assay was performed to explore how alteredhistone acetylation resulting from apelin-13 treatmentaffects the transcriptional level of Icam-1. Four sets ofprimers were designed to cover sequences containing theNF-κB binding site on the promoter of Icam-1. In Akitamice, ac-K significantly bound to the p1 sequence, andapelin-13 markedly inhibited this binding (Fig. 4E). Atthe same time, the phosphorylation level of p65 (activatedform of transcriptional subunit of NF-κB), but not thetotal level of p65, was significantly increased in the kidneyof Akita mice compared to WT mice (1.7-fold; Fig. 4Fand G). Apelin-13 suppressed diabetes-induced increasein p65 phosphorylation of Akita mice (85% reduction;Fig. 4F and G).

Apelin-13 protects mesangial cells from highglucose-induced inflammation by regulating histoneacetylation levels

To confirm the in vivo finding that apelin-13 mediateshistone deacetylation and reduction of inflammation, a ratmesangial cell line (HBZY-1) was used. The levels of totalac-K, ac-H3K9 and ac-H3K18 were markedly increased



in HBZY-1 cells cultured under a diabetic-like condition(HG). Apelin-13 reversed HG-induced histone hyper-acetylation in a dose-dependent manner (Fig. 5A and B).However, similar histone acetylation levels were found inthe NG cultured cells with or without 300 pM apelin-13(Supplemental Fig. S3A and B). The concentration(300 pM) of apelin used for the in vitro study is about5% of the circulation level of apelin (5.9 nM) after along-term injection of apelin to Akita mice (400 pmol (kgbody weight)–1), as we previously demonstrated using anELISA assay (Chen et al. 2011).

The immunofluorescence staining demonstrated thatthere were much fewer HDAC1-positive cells in the HGgroup compared to the NG group, while 300 pM apelin-13reversed the HG-induced decrease in HDAC1 (Fig. 5C).Western blotting also demonstrated that HDAC1 was

significantly decreased in the HG-cultured HBZY-1 cellscompared to the NG-cultured cells (43% decrease; Fig. 5Dand E), while apelin-13 normalized the HG-inducedreduction of HDAC1 level in a dose-dependent manner(Fig. 5D and E). On the other hand, GCN5 wassignificantly down-regulated in the HG group comparedto the NG group (Fig. 5F and G). However, the expressionlevels of HDAC2 and PCAF were unchanged among allexperimental groups (Fig. 5D–G). Furthermore, similarHDCA1/2 and PCAF/GCN5 levels were found in theNG incubated cells with or without 300 pM apelin-13(Supplemental Fig. S3C and D).

The transcription of inflammatory genes was furtherinvestigated in the HBZY-1 cells. Real-time PCR analysisrevealed that Mcp-1, Icam-1 and Vcam-1 mRNA levelswere significantly increased in the HG cultured cells

Figure 2. Apelin-13 treatment inhibits diabetes-induced glomerular hypertrophy and mesangialexpansion in Akita miceRepresentative PASH staining pictures (A) and quantitative analysis of glomerular area (B) in different experimentalgroups. Representative collagen IV staining pictures (C) and quantitative analysis of collagen IV-stained area in theglomeruli (D) in different experimental groups. Bar = 50 μm. Brown colour indicates collagen IV-positive staining.WT, wild-type mice, n = 9; WT + Ap, apelin-13-treated wild-type mice, n = 10; AK, Akita mice, n = 9; AK + Ap,apelin-13-treated Akita mice, n = 11. ∗P < 0.05 compared to WT mice; #P < 0.05 compared to Akita mice.



compared to the NG group (1.8-fold, 1.6-fold and 3.7-foldincrease, respectively; Fig. 5H), while these HG-inducedelevations of inflammatory genes were inhibited byapelin-13 (Fig. 5H).

To test whether the observed effects of apelin-13 areonly specific for the HBZY-1 cells, a mouse mesangialcell line (MES13) was also tested. Consistent with theresults obtained in HBZY-1 cells, the levels of totalac-K, ac-H3K9 and ac-H3K18 were significantly increasedin the HG cultured MES13 cells compared to the NGgroup (2.0-fold, 32-fold, 83-fold increase, respectively;Fig. 6A and B), while apelin-13 inhibited HG-inducedhistone hyperacetylation in a dose-dependent manner(Fig. 6A and B). Furthermore, the protein levels ofHDAC1 and HDAC2 were markedly reduced in theHG group compared to the NG group (80% and 29%reduction, respectively; Fig. 6C and D); however, onlythe HG-induced reduction of HDAC1 was reversed byapelin-13 (Fig. 6C and D). Furthermore, similar histoneacetylation levels, HDCA1/2 and PCAF/GCN5 levels were

found in the NG-treated MES13 cells with or without300 pM apelin-13 (Supplemental Fig. S4A–D).

Similar to the HBZY-1 cells, the mRNA levels ofMcp-1, Icam-1 and Vcam-1 were all significantly increasedin the HG group in MES13 cells (1.3-fold, 3.2-fold,1.7-fold increase, respectively; Fig. 7A), whereas 300 pM

apelin-13 significantly inhibited HG-induced increasesin these inflammatory genes (Fig. 7A). Further, thephosphorylation level of p65, but not total protein level,was significantly increased in the HG group (3.2-foldand 2.1-fold increase, respectively; Fig. 7B and C),while apelin-13 inhibited the HG-induced increase inphosphorylated p65 in a dose-dependent manner.

Apelin-13 protects mesangial cells from NaB-inducedinflammation by regulating histone acetylation levels

To further investigate the effects of apelin-13 on otherstimuli that can initiate histone hyperacetylation, NaBwas used to treat HBZY-1 cells. Compared to untreated

Figure 3. Apelin-13 inhibits diabetes-induced histone hyperacetylation by up-regulating HDAC1 levelin renal cortexRepresentative Western blots of ac-K, ac-H3K9, ac-H3K18, ac-H3K23, H3, HDAC1, HDAC2, GCN5, PCAF andβ-actin were shown in A, C and E, with densitometric quantitative analysis summarized in B, D and F. WT,wild-type mice, n = 4; AK, Akita mice, n = 5; AK+Ap, apelin-13-treated Akita mice, n = 5. ∗P < 0.05 comparedto WT mice; #P < 0.05 compared to Akita mice.



cells, NaB induced a significant decrease in the numberof HDAC1-positive cells, while apelin-13 rescued theNaB-induced decrease in HDAC1 in a dose-dependentmanner (Fig. 8A). Western blotting also demonstrated thatHDAC1 was dramatically decreased after NaB treatment(2.3-fold reduction), whereas 300 pM apelin-13 inhibitedthe NaB-induced a reduction of HDAC1 (Fig. 8B and C).However, at the doses used, apelin-13 showed no effecton NaB-induced down-regulation of HDAC2 (Fig. 8Band C).

In parallel with the decreased HDACs levels, thelevels of total ac-K, ac-H3K9 and ac-H3K18 weresignificantly increased after NaB treatment compared tothe untreated cells (31-fold, 11-fold and 19-fold increase,respectively; Fig. 8D and E). Treating cells with 300 pM

apelin-13 markedly reduced the NaB-induced histone

hyperacetylation (Fig. 8D and E). The mRNA levelsof Mcp-1, Icam-1 and Vcam-1 were also significantlyincreased in the NaB-treated cells (1.7-fold, 1.3-fold and3.1-fold increase, respectively), while the NaB-inducedupregulation of inflammatory gene was significantly lowerafter apelin-13 treatment (Fig. 8F).

Apelin directly alters histone acetylation levels inmesangial cells

To confirm the direct effect of apelin on histone acetyl-ation, HBZY-1 cells were transfected with either acontrol β-gal plasmid or an apelin expression plasmid.Successful transfection was demonstrated by a dramaticoverexpression of apelin mRNA in cells transfected withthe apelin plasmid compared to those transfected with

Figure 4. Apelin-13 suppressesdiabetes-induced inflammatory responsein the kidneys of Akita miceRepresentative Western blots of MCP-1,ICAM-1, iNOS and β-actin (A) withsummarized densitometric quantitative analysis(B). Representative F4/80 staining pictures (C)and quantitative analysis of F4/80-stainedpositive cells in the glomeruli (D) in differentexperimental groups. Bar = 100 μm. Browncolour indicates F4/80 positive staining. E,quantitative analysis of PCR of chromatinimmunoprecipitated DNA which measure thebinding affinities of ac-K to the promoters ofIcam-1. The abundance is relative to the inputin the same sample with the same primer. F,representative Western blots of p-p65, p65and β-actin; G, summarized densitometricquantitative analysis. WT, wild-type mice,n = 4; AK, Akita mice, n = 5; AK+Ap,apelin-13-treated Akita mice, n = 5. ∗P < 0.05compared to WT mice; #P < 0.05 compared toAkita mice.



Figure 5. Apelin-13 inhibits the highglucose-induced hyperacetylationthrough normalization of HDAC1 inHBZY-1 cellsRepresentative Western blots of ac-K,ac-H3K9, ac-H3K18 and H3 (A) withdensitometric quantitative analysis (B). C,representative HDAC1 stained pictures fordifferent experimental groups. Top panels,HDAC1 staining (red); bottom panels, DAPIstaining (blue). Bar = 50 μm D–G,representative Western blots of HDAC1,HDAC2, GCN5, PCAF and β-actin (D andF), with summarized densitometricquantitative analysis (E and G). H, qRT-PCRanalysis of Mcp-1, Icam-1 and Vcam-1levels in different experimental groups.Each experiment was performed intriplicate and repeated for three times. Arepresentive result was shown. NG, 5.5 mM

glucose; HG, 25 mM glucose; HG + 30 pM

Ap, 25 mM glucose with 30 pmol l−1

apelin-13; HG + 300 pM Ap, 25 mM

glucose with 300 pmol l−1 apelin-13.∗P < 0.05 compared to NG group;#P < 0.05 compared to HG group.

the control plasmid (Fig. 9A). The transferred cells werecultured in either NG or HG media. Compared to theHG cultured cells transfected with the control β-galplasmid, overexpression of apelin markedly suppressedthe HG-induced upregulation of ac-H3K9 and ac-H3K18(Fig. 9B and C).

Discussion

Approximately 50% of diabetic patients developnephropathy, which makes DN the leading cause ofchronic kidney disease in most Western countries andChina (Villeneuve et al. 2011; Vlassara et al. 2012; Xuet al. 2013). New therapies are in great demand. The



ability of apelin to lower blood pressure has been welldocumented (Zhao et al. 2010; Akcilar et al. 2013), whichmakes it a good candidate for DN treatment. A recent studysuggested that subcutaneous administration of apelin-13reduced kidney and glomerular hypertrophy as well asrenal inflammation without affecting blood pressure indiabetic FVB/Ove26 mice (Day et al. 2013). We also foundthat apelin-13 significantly inhibited diabetes-inducedrenal dysfunction, in specific, glomerular hypertrophy andtype IV collagen accumulation in Akita mice. Both studiesshowed the benefit of using apelin-13 to treat DN in animalmodels of type 1 diabetes.

Apelin and its receptor, APJ, are widely expressed in avariety of tissues, including adipose tissue, brain, lung andkidney (Medhurst et al. 2003; Kleinz & Baxter, 2008). Inrenal diseases, the expression levels of apelin and APJ in thekidneys are altered. In the 2K1C hypertension rat model,

the mRNA and protein levels of the renal apelin and APJwere both reduced (Najafipour et al. 2012), which maybe responsible for the dysfunction in vascular tone in thekidney. We further demonstrated that the level of apelin,the endogenous ligand of APJ, was also down-regulated inthe kidneys of diabetic mice.

Histone hyperacetylation has been reported in severalanimal models of renal diseases, including DN. In db/dbmice with uninephrectomization, the H3K9 and H3K23acetylation levels are significantly increased compare tothose of WT mice undergoing the same surgery. Inthe kidneys of type 1 diabetic rats, increased bindingaffinity of acetylated histone H3 to the promoter of theFbn1 gene, which encoded an extracellular matrix-protein,fibrillin 1 was found (Gaikwad et al. 2010). Furthermore,increased histone acetylation at the promoters of Mcp-1(proinflammatory) and Collagen III (profibrotic) genes

Figure 6. Apelin-13 inhibits the high glucose-induced histone hyperacetylation by up-regluratingHDAC1 in MES 13 cellsRepresentative Western blot analysis of ac-K, ac-H3K9, ac-H3K18, H3, HDAC1, HDAC2, GCN5, PCAF and β-actinwere shown in A, C and E, with densitometric quantitative analysis summarized in B, D and F. Each experimentwas performed in triplicate and repeated for three times. A representive result was shown. NG, 5.5 mM glucose;HG, 25 mM glucose; HG + 30 pM Ap, 25 mM glucose with 30 pmol l−1 apelin-13; HG + 300 pM Ap, 25 mM

glucose with 300 pmol l−1 apelin-13. ∗P < 0.05 compared to NG group; #P < 0.05 compared to HG group.



has been demonstrated after acute kidney injury andis associated with significant overexpression of thesetwo genes (Zager et al. 2011). Furthermore, increasedrecruitment of ac-H3K9 and NF-κB are found on thepromoters of TNFα and COX-2 in HG-treated monocytes,suggesting that histone acetylation leads to circulatoryinflammation in diabetes (Miao et al. 2004; Pero et al.2011). Our data also showed that an antibody against ac-Ksignificantly bound to the p1 sequence of Icam-1 promoter,whereas apelin-13 treatment reversed this increase in Akitamice. In the present study, administration of apelin-13markedly suppressed diabetes or HG-induced histonehyperacetylation in both in vivo and in vitro models.Furthermore, over-expression of apelin in mesangial cellsinhibited high glucose-induced upregulation of ac-H3K9and ac-H3K18. Thus, we demonstrated that apelin per secan regulate histone acetylation levels.

Interestingly, among all tested HATs (PCAF andGCN5) and HDACs (HDAC1 and HDAC2), apelin-13selectively regulated the expression of HDAC1. Therole of HDACs in the pathogenesis of DN is stillcontroversial. Long-term administration of vorinostat, anHDAC inhibitor, attenuates renal injury in STZ-induceddiabetic mice (Advani et al. 2011). Inhibition of HDAC-2,specifically, is beneficial for TGF-β-induced renal injury

(Noh et al. 2009). However, inhibition of HDACsmay inhibit the increased expression of diabetes-inducedinflammatory genes (Edelstein et al. 2005; Fish et al. 2005;Reddy et al. 2009).

Elevated inflammatory molecules, such as adhesionmolecules and chemokines, have been reported tocontribute to the pathogenesis of DN. ICAM-1 andVCAM-1 enable leucocytes to adhere to endotheliumand to migrate into inflammatory sites, which eventuallyinitiate inflammatory responses (Alkhalaf et al. 2012).The important role of ICAM-1 in the development of DNhas been clearly demonstrated in ICAM-1-deficient db/dbmice, which were generated by crossing ICAM-1-deficientmice with db/db mice, a well-known type 2 diabeticmodel. Diabetes-induced glomerular hypertrophy, hyper-cellularity and tubular damage are all reduced inICAM-1-deficient db/db mice (Chow et al. 2005). MCP-1,a chemokine, promotes monocyte infiltration and leads tothe accumulation of inflammatory cells in organs like thekidney (Chow et al. 2007). In DN patients, CCR2 (thereceptor of MCP-1) is overexpressed in the glomerularpodocytes while in streptozotocin (STZ)-induced diabeticmice, MCP-1 is overexpressed in glomeruli (Tarabraet al. 2009). Furthermore, MCP-1 deficiency reducesdiabetes-induced albuminuria both in a type 1 diabetes

Figure 7. Apelin-13 inhibits the high glucose-induced elevation of inflammation in MES 13 cellsA, qRT-PCR analysis of Mcp-1, Icam-1 and Vcam-1 levels in different experimental groups. B, representativeWestern blots of p-p65, p65 and β-actin; C, summarized densitometric quantitative analysis. Each experiment wasperformed in triplicate and repeated for three times. A representative result was shown. NG, 5.5 mM glucose; HG,25 mM glucose; HG + 30 pM Ap, 25 mM glucose with 30 pmol l−1 apelin-13; HG + 300 pM Ap, 25 mM glucosewith 300 pmol l−1 apelin-13. ∗P < 0.05 compared to NG group; #P < 0.05 compared to HG group.



Figure 8. Apelin-13 suppresses histone acetylation by up-regulating HDAC1 in HBZY-1 cells culturedwith HDACs inhibitorA, representative HDAC1 stained pictures for different experimental groups. Top panels, HDAC1 staining (redcolour); bottom panels, DAPI staining (blue colour). Bar = 50 μm. B and D, representative Western blots of HDAC1,HDAC2, ac-K, ac-H3K9, ac-H3K18, H3, and β-actin; C and E, summarized densitometric quantitative analysis. F,qRT-PCR analysis of Mcp-1, Icam-1 and Vcam-1 levels in different experimental groups. CT; normal media; NaB,normal media with 5 mM NaB; NaB + 30 pM Ap, 5 mM NaB with 30 pmol l−1 apelin-13; NaB + 300 pM Ap, 5 mM

NaB with 300 pmol l−1 apelin-13. Each experiment was performed in triplicates for three times. A representativeresult was shown. ∗P < 0.05 compared to CT group; #P < 0.05 compared to NaB group.



model and a type 2 diabetes model (Chow et al.2007; Tarabra et al. 2009), which makes it an attractivetherapeutic target for DN. In fact, a recent study reportedthat an MCP-1 antagonist, mNOX-E36, significantlyinhibits diabetes-induced glomerulosclerosis in db/dbmice (Sayyed et al. 2010). In this study, we showed thatapelin-13 significantly suppressed several inflammatorymolecules, including MCP-1, ICAM-1, VCAM-1 andiNOS, in the renal cortex of Akita mice, as well as inhigh glucose-cultured mesangial cells. We also found thatapelin-13 treatment greatly inhibited diabetes-inducedinfiltration by macrophages (F4/80-positive cells) in Akitamice.

The effects of apelin-13 in vivo and in vitro have beenalso investigated in normal conditions. In WT mice,administration of apelin-13 caused neither change inmorphology such as glomerular area and mesangial matrixarea, nor changes in some renal functions like kidneyweights and A/G; however, apelin treatment showed asignificant effect on proteinuria and GFR. Significantreduction of GFR below normal levels is usually associatedwith loss of nephrons and renal function(Foundation,2002). However, the number of glomeruli is similarbetween WT mice treated with or without apelin-13 (datanot shown). There are some factors such as blood pressurelevel, extracellular fluid volume and the availability ofthe secreted tubular creatinine that affect the final resultof GFR (Foundation, 2002). Furthermore, there are no

obvious changes in histone acetylation levels and theenzymes regulating histone acetylation levels (ones weexamined) of WT mice and NG cultured mesangial cellswhen treated with apelin-13. We thus proposed thatthe administration of apelin-13 would not affect therenal function in normal conditions, although the effectof apelin-13 on GFR in the WT mice needs furtherinvestigation.

Figure 10. The potential mechanisms of the inhibitory effectsof apelin-13 on diabetic nephropathy

Figure 9. Overexpression of apelin suppresses histone acetylation in HBZY-1 cells cultured with highglucoseA, RT-PCR analysis of Apln and 18S rRNA levels in HBZY-1 cells. Apelin or pPCAGSIH-β-gal plasmid was transfectedin HBZY-1 and the efficiency of overexpression was assessed. B, representative Western blots of ac-H3K9, ac-H3K18and H3; C, summarized densitometric quantitative analysis. Each experiment was performed in triplicates for threetimes. A representative result was shown. CT; normal media transfected with the pPCAGSIH-β-gal plasmid;HG + β-gal, 25 mM glucose media transfected with the pPCAGSIH-β-gal plasmid; HG + apelin, 25 mM glucosemedia transfected with the pPCAGSIH-apelin plasmid. ∗P < 0.05 compared to CT group; #P < 0.05 compared toHG + β-gal group.



In summary, we have demonstrated that apelin-13at a low dose significantly inhibited diabetes-inducedinflammation, renal hypotrophy and glomerularexpansion in Akita mice. We further demonstratedthat increased histone acetylation and alteration ofcorresponding histone acetylation modifying enzymesin the renal cortex of Akita mice and high glucosecultured mesangial cells are associated with inflammation(Fig. 10). We show, for the first time, that apelin-13treatment not only inhibits diabetes-induced elevatedhistone acetylation, but also up-regulates HDAC1 both invivo and in vitro (Fig. 10). Thus, apelin may be a noveltherapeutic candidate for treatment of DN via regulatinghistone acetylation.

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Additional information

Competing interests

The authors declare no competing interests.



Author contributions

L.Z. and K.H. designed the experiments; H.C., J.S.L., J.L., A.P.and L.H.J. performed the experiments; H.C., R.B.P., L.Z. andK.H. analysed the data; H.C., R.B.P., L.Z. and K.H. wrote themanuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Basic ResearchProgram of China (2012CB524901), the Natural ScienceFoundation of China (Nos 31271370, 81100687, 81172971 and

81222043), the Program for New Century Excellent Talents inUniversity (NECT10-0623 and NECT11-0170), the MunicipleKey Technology Program of Wuhan (Wuhan Bureau of Science& Technology, No. 201260523174), the Health Bureau of Wuhan(WX12B06) and the Natural Science Foundation of HubeiProvince (2013CFB359).

Acknowledgements

We thank Dr Takakura (Osaka University) for apelin plasmid.We also thank Mr Hao Gong and Miss Lianqi Huang for technicalassistance.


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