BBADIS-64174; No. of pages: 10; 4C: 3, 5, 6 Biochimica et Biophysica Acta xxx (2015) xxx–xxx

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Article history: Received 28 October 2014 Received in revised form 12 February 2015 Accepted 27 February 2015 Available online xxxx

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Keywords: Apelin Renal I/R injury Histone methylation TGF-β1

Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan, China, 430030 Centre for Biomedicine Research, Wuhan Institute of Biotechnology, Wuhan, China, 430074 College of Life Sciences, Wuhan University, Wuhan, China, 430072 d Wuhan the Third Hospital, Wuhan, China, 430060 e Department of Pathology, Case Western Reserve University, Cleveland, OH, USA, 44106 f Department of Neuroscience, Case Western Reserve University, Cleveland, OH, USA, 44106 g Department of Neurology, Case Western Reserve University, Cleveland, OH, USA, 44106 b

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Renal ischemia/reperfusion (I/R) injury is the most common cause of acute kidney injury, having a high rate of mortality and no effective therapy currently available. Apelin-13, a bioactive peptide, has been shown to inhibit the early lesions of diabetic nephropathy in several mouse models by us and others. To test whether apelin-13 protects against renal I/R induced injury, male rats were exposed to renal I/R injury with or without apelin-13 treatment for 3 days. Apelin-13 treatment markedly reduced the injury-induced tubular lesions, renal cell apoptosis, and normalized the injury induced renal dysfunction. Apelin-13 treatment inhibited the injury-induced elevation of inflammatory factors and Tgf-β1, as well as apoptosis. Apelin-13 treatment also inhibited the injury-induced elevation of histone methylation and Kmt2d, a histone methyltransferase of H3K4me2, following renal I/R injury. Furthermore, in cultured renal mesangial and tubular cells, apelin-13 suppressed the injuryinduced elevation of Tgf-β1, apoptosis, H3K4me2 and Kmt2d under the in vitro hypoxia/reperfusion (H/R) conditions. Consistently, over-expression of apelin significantly inhibited H/R-induced elevation of TGF-β1, apoptosis, H3K4me2 and Kmt2d. The present study therefore suggests apelin-13 may be a therapeutic candidate for treating acute kidney injury. © 2015 Published by Elsevier B.V.

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1. Introduction

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Renal ischemia/reperfusion (I/R) injury is the most common cause of acute kidney injury (AKI) after major surgery or renal transplantation in both allograft and native kidneys [1]. AKI affects 5% of all hospitalized patients with an unacceptably high rate of mortality [2]. AKI always implies a poor prognosis with no effective therapy currently available [3]. Renal I/R injury usually causes primary tubular epithelial cell injury, including tubular obstruction and reduced tubular re-absorption of NaCl. However, several studies reported that hemodynamic changes induced

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Hong Chen a,b, Danyang Wan c, Lin Wang c, Anlin Peng d, Hongdou Xiao c, Robert B. Petersen e,f,g, Chengyu Liu a,b, Ling Zheng c,⁎, Kun Huang a,b,⁎⁎

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Apelin protects against acute renal injury by inhibiting TGF-β1

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Abbreviations: AKI, Acute kidney injury; ATN, Acute tubular necrosis; TGF-β1, Transforming growth factor β1; HAT, Histone acetyltransferase; HDAC, Histonedeacetylase; HMTs, Histone methyltransferases; HDMs, Histone demethyltransferases; H/R, Hypoxia/reperfusion; I/R, Ischemia/reperfusion; ICAM-1, Intercellular adhesion molecule 1; PARP-1, Poly(ADP-ribose) polymerase-1; MCP-1, Monocyte chemotactic protein 1; 2K1C, Two-kidney-one-clip ⁎ Corresponding author. ⁎⁎ Correspondence to: K. Huang, Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan, China, 430030. E-mail addresses: [email protected] (L. Zheng), [email protected] (K. Huang).

by renal I/R injury also occur, which lead to mesangial contraction and cessation of glomerular filtration. Thus, the interaction of microvascular (including glomerular and medullary parts) and tubular events both contribute to the pathogenesis of acute ischemic renal failure [4,5]. Although renal I/R injury has been extensively studied, the pathophysiologic process involving inflammation and cell apoptosis, as well as its relationship to the subsequent renal injury, remain to be fully elucidated. TGF-β1 (transforming growth factor-β1), a key member of TGF super-family, has been established as the central mediator of renal fibrosis and inflammation associated with multiple progressive kidney diseases [6,7]. Fibrosis represents the final step of renal injury which ultimately leads to end-stage kidney failure [8], therefore, blocking TGF-β1 is an attractive approach to prevent renal injury. Histone modifications, including acetylation, methylation and phosphorylation, play important roles in the regulation of transcriptional activity [9–12]. Histone acetylation, which is mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), regulates chromatin structure and controls genes expression [13]. Recent studies have shown that changes in the levels of HDACs are associated with renal I/R injury [14,15]. In addition, histone methyltransferases

http://dx.doi.org/10.1016/j.bbadis.2015.02.013 0925-4439/© 2015 Published by Elsevier B.V.

Please cite this article as: H. Chen, et al., Apelin protects against acute renal injury by inhibiting TGF-β1, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbadis.2015.02.013

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Male Wistar rats were obtained from the Hubei Animal Laboratory, and housed in ventilated microisolator cages with free access to water and food. Rats weighing 180 ± 20 g were used and assigned to one of the following groups: CT group, uninjured rats with vehicle administration; I/R group, rats underwent I/R injury with vehicle administration; I/R + Ap group, rats underwent I/R injury with 5 μg/kg BW apelin-13 administered twice per day. Apelin-13 was chemically synthesized by standard Fmoc strategy and purified to N98% with rp-HPLC as we previously described [32,33]. I/R injury was performed as previously described [34]. Briefly, rats were anesthetized and underwent midline abdominal incisions with their left renal pedicle bluntly clamped by a clamp for 30 min (unilateral renal occlusion). After removing the clamps, wounds were sutured and the animals were allowed to recover for 3 days before sacrifice. Control animals were sham operated. All animal experiments were approved by the Committee on Ethics in the Care and Use of Laboratory Animals of the College of Life Sciences, Wuhan University.

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2.2. Assessment of renal function

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Twenty-four-hour urine samples were collected in metabolic cages 1 day before sacrifice. Urine levels of creatinine and total protein were measured with an Olympus AU2700 automatic biochemistry analyzer using a creatinine reagent kit (Fuxing Changzheng Medical Inc., Shanghai, China) or a total protein reagent kit (Greatwall Clinical Reagent Inc., Baoding, China) as we previously described [24].

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2.4. TUNEL assay

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Paraffin-embedded sections were deparaffinized and rehydrated as previously reported [24]. Apoptotic cells were detected by TUNEL assay using an In Situ Cell Death Detection Kit (Roche, Mannheim, Germany) as previously described [36]. At least six different areas per renal sample of TUNEL positive cells were counted using an Olympus BX60 microscope equipped with a digital CCD and reported as number of TUNEL-positive nuclei divided by the total number of cells per field.

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2.5. Cell culture and in vitro H/R injury

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Renal sections were stained with hematoxylin and eosin (H&E). Hallmarks of acute tubular necrosis (ATN) were examined on a double-blind basis. The degree of renal damage was semi-quantitatively evaluated with a scale in which 0 represents no abnormalities, and 1+, 2+, 3+, 4+ stand for slight (up to 20%), moderate (20 to 40%), severe (40 to 60%), and total necrosis (affecting more than 80% of renal parenchyma), respectively [35].

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2.3. Renal histology

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HBZY-1 (a rat mesangial cell line) and NRK-52E (a rat tubular epithelia cell line) were cultured in DMEM media (Hyclone, South Logan, USA) with 5.5 mM glucose plus 5% fetal bovine serum (FBS) (Gibco, Grand Island, USA), and maintained in a 37 °C incubator (Thermo scientific, Marietta, USA) with humidified atmosphere of 21% O2 which was regarded as the normal culture condition. In vitro H/R experiments were performed as previously described with slight modifications [37]. At 80% confluence, cells were put into a 37 °C incubator under 1% O2 with the normal media replaced with media lacking glucose and FBS, which was regarded as hypoxia. After culturing under the hypoxia condition for a specified amount of time (4 hrs for HBZY-1 cells and 1 hr for NRK-52E cells), cells were returned to the normal culture condition for two hours, which was regarded as reperfusion. For apelin-13 treatment, 300 pM apelin-13 was added to media during the hypoxia and reperfusion period as in our previous report [24]. 2-TCP (trans-2Phenylcyclopropylamine hydrochloride), a HMT inhibitor [38], was added to media to induce the total methylation of H3K4 in the cultured renal cells.

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2.6. Transfection

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HBZY-1 cells were plated in six-well plates and transfected the next day with either pPCAGSIH-β-gal or pPCAGSIH-apelin plasmids (kind gifts from Dr. Takakura, Osaka University) using Fugene HD transfection reagent (Promega, Madison, USA) according to the manufacturer's instruction.

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2.7. Western blots

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Freshly collected kidney or cultured cells were sonicated in ice-cold RIPA buffer (Beyotime, Haimen, China) and protein concentrations were quantitated as previously described [39]. 20–80 μg of protein from each sample were separated by SDS-PAGE. The proteins were transferred onto PVDF membranes for immunodetection. The list of antibodies used in the present study is provided in Supplementary Table S1. The intensity of the targeted protein bands was evaluated using Quantity One 1-D Analysis Software. Individual protein levels were quantitated relative to the β-actin level in the same sample and further normalized to the respective control group, which was set at one.

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(HMTs) and histone demethyltransferases (HDMs) play critical roles in chromatin remodeling and gene expression [16]. However, the roles of histone methylation or enzymes that regulate histone methylation in the pathogenesis of acute renal I/R injury remain unknown. The apelin family of adipokines is derived from a 77-residue preproprotein with lengths varying from 12 to 36 residues [17,18]. Apelin-13, the most active member of the apelin group, has multiple biological functions including regulation of glucose balance, blood pressure, cardiovascular functions, blood vessel integrity and renal function [19–24]. APJ (angiotensin I receptor-related protein J receptor), which belongs to GPCR (G-protein-coupled receptor) family, is the only endogenous receptor for apelin. Apelin and APJ are widely expressed in a variety of tissues like kidney, adipose, brain and lung [25,26]. Several animal models indicate that the apelin–APJ system plays a critical role in the regulation of cardiovascular fluid homeostasis [27,28]. The apelinergic system (apelin and its receptor APJ) has also been suggested to be a promising therapeutic target in obesity-associated insulin resistance [29]. Recently, we and others demonstrated that administration of apelin13 reduces kidney and glomerular hypertrophy as well as renal inflammation in type 1 diabetic mice [24,30], which makes apelin-13 a good candidate for treating diabetic nephropathy [31]. Based on this finding, we wondered whether apelin-13 might also have beneficial effects on acute renal injury, such as renal I/R injury. In the present study, we administered apelin-13 to renal I/R injured rats. The injury-induced tubular lesions, renal dysfunction, renal cell apoptosis, alterations in histone methylation and histone methyltransferase/demethylase, as well as the levels of Tgf-β1 were analyzed. In addition, in vitro hypoxia/reperfusion (H/R) injury was performed on cultured renal mesangial cells and tubular cells, and the effects of administering apelin-13 and apelin over-expression on the injury-induced apoptosis, elevation of Tgf-β1, alterations in histone methylation and HMTs/HDMs were assessed. Apelin-13 was found to suppress renal inflammation and apoptosis in both I/R or H/R injury by inhibiting Tgf-β1. These results suggest that apelin-13 should be considered as a new therapeutic target for the treatment of AKI.

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Please cite this article as: H. Chen, et al., Apelin protects against acute renal injury by inhibiting TGF-β1, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbadis.2015.02.013

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Kidneys were cross-linked with 1% formaldehyde for 10 min and a ChIP assay was performed as previously described [24]. Chromatin was immunoprecipitated with an anti-H3K4me2 antibody (Abcam, Cambridge, UK). The purified DNA was detected by PCR. The primer sequences used are provided in supplementary Table S1. The input samples were used as an internal control for comparison between samples.

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2.10. Statistical analysis

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The data were expressed as mean ± standard error of the mean (SEM). Statistical significance was determined by analyzing the data with the nonparametric Kruskal–Wallis test, followed by the Mann– Whitney test. Differences were considered statistically significant at P b 0.05.

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3. Results

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3.1. Apelin-13 protects against renal I/R injury induced morphological and functional changes

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The effects of different dosages of apelin-13 (1, 2, 5 and 10 μg/kg BW) on the renal morphological changes were first examined at 3 days after I/R injury (Fig. S1). Compared to non-injured kidneys, I/R injury led to severe tubular damage, as evidenced by widespread tubular

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3.2. Apelin-13 normalizes I/R or H/R injury induced down-regulation of 236 Apln mRNA level in vivo and in vitro 237 The endogenous Apln mRNA level was examined in different experimental groups. As expected, the mRNA level of Apln was significantly decreased in the kidney after I/R injury, while this injury-induced decrease was largely prevented by administration of apelin-13 (Fig. 2A). At the same time, the protein level of APJ, the endogenous receptor for apelin, was similar among experimental groups. Hypoxia inducible factor 1α (Hif1α), a key factor involved in the cellular and systemic responses to hypoxia, has been demonstrated to contribute to the pathogenesis of I/R injury [40]. The protein level of Hif1α was increased in the kidneys after I/R injury, whereas administration of apelin-13 significantly reduced I/R injury-induced up-regulation of Hif1α (Fig. 2B). Two different renal cell lines were used to evaluate the effects of apelin-13 treatment under in vitro H/R condition. A known hypoxia sensor, the levels of Hif1α markedly increased after H/R injury in both NRK-52E cells and HBZY-1 cells (Fig. 2D and F), indicating the successful establishment of hypoxic injury in these cells. In NRK-52E cells, H/R led to a significant decrease in Apln mRNA level. The injury-induced decrease in Apln mRNA was significantly reduced by administrating apelin-13 to the injured renal tubular epithelial cells (Fig. 2C). There was no significant change in the APJ protein level among the

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Total RNA was isolated from kidneys or cultured cells using RNAiso Plus (TaKaRa Biotechnology, Dalian, China). Total RNA was reverse transcribed into cDNA using M-MLV first strand synthesis system (Invitrogen, Grand Island, USA). The abundance of specific gene transcripts was assessed by PCR. The sequences of the primers used are listed in Supplementary Table S2. PCR products were separated by electrophoresis using 1.5% agarose gels, and images were photographed using a Molecular Imager Gel Doc XR (Bio-Rad, Hercules, USA). Band density was quantified using Quantity One 1-D Analysis Software (Bio-Rad). The mRNA level of the targeted gene was quantitated first against the Rn18s level from the same sample, then normalized to the non-injured group or the cells transferred with pPCAGSIH-β-gal under normal conditions, which was set at one.

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necrosis, cast formation and tubular cells denudation (Figs. 1A and S1). Histochemistry results demonstrated that administration of 5 and 10 μg/kg BW of apelin-13 has protective effects on renal morphology, while lower doses (1 or 2 μg/kg BW) showed no obvious beneficial effects on renal morphology after the I/R injury (Fig. S1). Semiquantitative results demonstrated that a 5 μg/kg apelin-13 treatment significantly improved the pathological scores in the injured kidneys (Fig. 1B); this dose was thus chosen for further in vivo experiments. Kidney damage was assessed by measurement of 24-h urine volume, proteinuria and urine creatinine. Functional renal studies demonstrated that renal I/R injury caused kidney dysfunction, as reflected by significantly elevated 24-h urine volume, proteinuria and urine creatinine (Fig. 1C–E); whereas apelin-13 treatment suppressed I/R injuryinduced elevation of these renal function parameters (Fig. 1C–E). However, the calculated ratio of protein/creatinine in different experiment groups suggested that 3 days after I/R-injury, there was no increase in the protein/creatinine ratio, nor did apelin show any effect on this ratio (Supplementary Fig. S2).

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Fig. 1. Apelin-13 treatment inhibits I/R injury induced renal dysfunction. Representative HE staining pictures (A) and quantitative results of renal damage score (B) in different experimental groups. (C) Urine volume in different experimental groups. (D) Proteinuria in different experimental groups. (E) Urine creatinine concentration in different experimental groups. CT, non-injured rats; I/R, I/R injured rats; I/R + Ap, apelin-13 treated I/R injured rats, n = 5–7 per group. § indicates vast formation; arrow indicates tubular cells denudation; € indicates tubular necrosis. ⁎p b 0.05 compared to CT rats; #p b 0.05 compared to I/R injured rats.

Please cite this article as: H. Chen, et al., Apelin protects against acute renal injury by inhibiting TGF-β1, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbadis.2015.02.013

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3.3. Apelin-13 suppresses I/R or H/R injury induced inflammation and apoptosis in kidneys and cultured renal cells

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Up-regulation of inflammatory chemokines and adhesion molecules in the injured sites causes inflammatory cells to infiltrate, which in turn amplifies I/R injury [41,42]. We observed that the level of MCP-1, an inflammatory chemokine, and the level of ICAM-1, an adhesion molecule, were both significantly induced in the kidneys after renal I/R injury. Apelin-13 treatment significantly reduced the injury-induced upregulation of ICAM-1 and MCP-1 in the kidneys (Fig. 3A). Cleavage of caspase-3 and caspase-8 activates these two caspases and represents the execution stage of cell death [14,43]. Compared to the non-injured kidneys, elevation of the cleaved, active forms of caspase-3 and caspase-8 were found in the kidneys of I/R injured rats. Administration of apelin-13 markedly attenuated such injury-induced activation of caspases (Fig. 3B). The activation of caspase-12 after renal I/R injury was also reported in a previous study [44]. Compared to the non-injured kidneys, there was a trend toward elevation of cleaved forms of caspase-12 in the kidneys of I/R injured rats, while administration of apelin-13 significantly inhibited the cleavage of caspase12 compared to the I/R-injury per se (Fig. 3B). To further determine whether apelin-13 treatment inhibits I/R injury-induced apoptosis in the kidneys, a TUNEL assay was performed. Substantially increased numbers of TUNEL+ cells were evident in renal tubular cells after the

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experimental groups (Fig. 2D). Similar to what we found in NRK-52E cells, the Apln mRNA level was also significantly decreased under a H/R condition, which was markedly up-regulated by apelin-13 treatment in the injured HBZY-1 cells (Fig. 2E). There was no significant difference in the APJ expression level between the untreated and H/R stimulated cells, however, 300 pM apelin-13 treatment significantly increased the APJ level in HBZY-1 cells after H/R stimulation (Fig. 2F). Furthermore, apelin-13 treatment normalized the injury-induced elevation of Hif1α level in both cell lines (Fig. 2D and F).

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Fig. 2. Apelin-13 normalized I/R or H/R injury induced downregulation of Apln mRNA level and Hif1α overexpression in kidneys, cultured renal cells. Representative PCR analysis with densitometric quantitative results of Apln, Rn18s were shown in (A, C, E). Representative Western blot analysis with densitometric quantitative results of APJ, Hif1α and β-actin were shown in (B, D, F). CT or NC, non-injured rats or cells; I/R or H/R, I/R injured rats or H/R injured cells; I/R or H/R + Ap, apelin-13 treated I/R injured rats or H/R injured cells, n = 3–5 per group. ⁎p b 0.05 compared to CT or NC group; #p b 0.05 compared to I/R or H/R group.

injury, while apelin-13 treatment significantly abrogated such injuryinduced increase in cell death (Fig. 3C). Consistent with the in vivo studies, the levels of the active, cleaved forms of caspase-3 and caspase-8 were also significantly increased in NRK-52E cells in the H/R injury model (Fig. 3D). Apelin-13 treatment markedly suppressed H/R stimulated activation of cell death (caspase-8 and caspase-3) in cultured renal tubular epithelial cells (Fig. 3D). Consistent with the results of NRK-52E cells, the levels of active, cleaved forms of caspase-3, caspase-8 and caspase-12 were also significantly increased in HBZY-1 cells following H/R injury (Fig. 3E). Apelin-13 treatment markedly suppressed H/R stimulated activation of cell death in cultured mesangial cells (Fig. 3E).

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3.4. Apelin-13 normalizes I/R or H/R injury induced alteration in histone 302 modifications in kidneys and cultured renal cells 303 We previously demonstrated that histone acetylation is involved in the pathogenesis of diabetic nephropathy [24], in this study, we sought to determine whether histone acetylation has similar effects on I/R injured kidneys. The changes in histone acetylation after renal I/R injury were investigated. The levels of ac-K, ac-H3K9, ac-H3K18, ac-H3K23, ac-H3K56 and ac-H4K8 were significantly increased in the kidneys after I/R injury compared to those in the non-injured kidneys (Fig. S3). Apelin-13 treatment markedly suppressed I/R injury-induced increases in the levels of ac-K, ac-H3K23 and ac-H3K56 in the kidneys, and showed no significant effects on other histone acetylation sites (Fig. S3). However, no significant change to ac-K was found in H/R stimulated HBZY-1 and NRK-52E cells (data not shown). Furthermore, the levels of several histone methylation sites were examined. There were dramatic increases in the levels of H3K4me2 (4.7-fold), H3K9me3 (2.2-fold), H3K79me1 (5.9-fold) in the kidneys after I/R injury (Fig. 4A). On the other hand, the level of H4K20me3 was decreased in the I/R injury group (Fig. 4A), whereas the levels of H3K4me1 and H3K4me3 were not significantly changed in the kidneys

Please cite this article as: H. Chen, et al., Apelin protects against acute renal injury by inhibiting TGF-β1, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbadis.2015.02.013

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Fig. 3. Apelin-13 suppresses I/R or H/R injury induced inflammation and apoptosis in kidneys, cultured renal cells. Representative Western blot analysis with densitometric quantitative results of MCP-1, ICAM-1 and β-actin were shown in (A). Representative Western blot analysis with densitometric quantitative results of caspase 3 (35 KD), c-cas3 (19 KD), c-cas3 (17 KD), c-cas8 (26KD), c-cas8 (18KD), caspase12 (42 KD), c-cas12 (38 KD), and β-actin are shown in (B, D and E). (C) Representative TUNEL stained pictures for different experimental groups. Top panels, DAPI staining (blue color); bottom panels, TUNEL staining (green color). CT or NC, non-injured rats or cells; I/R or H/R, I/R injured rats or H/R injured cells; I/R or H/R + Ap, apelin13 treated I/R injured rats or H/R injured cells, n = 3–7 per group. ⁎p b 0.05 compared to CT or NC group; #p b 0.05 compared to I/R or H/R group.

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after I/R injury. Administration of apelin-13 significantly reduced the levels of H3K4me2 and H3K79me1 after I/R injury (Fig. 4A). The mRNA level of several HMTs (Ash1, Kmt2d, Set7/9, Set1, Dot1l) and HDMs (Kdm1a, Kdm5a) were further examined. Significant increases in the levels of Ash1, Kmt2d, Set7/9 and Dot1l were observed in the kidneys of I/R injured rats compared with those in the non-injured rats. Apelin-13 treatment suppressed I/R injury-induced up-regulation of Ash1, Kmt2d and Set7/9, but showed no effect on the level of Dot1l in the kidneys (Fig. 4B). There were no significant changes in the mRNA level of Set1, Kdm1a and Kdm5a among experimental groups (Fig. 4B). Furthermore, an immunochemical study of renal sections demonstrated that the level of Kmt2d was increased in renal tubules and glomeruli after I/R injury, and administration of apelin-13 significantly inhibited the injury-induced elevation of Kmt2d (Fig. 4C). To further confirm the in vivo finding that apelin-13 mediates histone methylation, we analyzed the levels of H3K4me2 and H3K79me1

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in the NRK-52E cells cultured under normal or H/R conditions. The levels of H3K4me2 and H3K79me1 were markedly increased in renal tubular epithelial cells cultured under H/R condition compared to those in control cells, whereas apelin-13 treatment only significantly abrogated the H/R-induced up-regulation of H3K4me2 (Fig. 4D). Moreover, substantial increases in H3K4me2 and H3K79me1 were evident in HBZY-1 cells cultured under H/R condition compared with those in controls, and such increases were reversed by apelin-13 treatment (Fig. 4F). Moreover, NRK-52E cells cultured under H/R condition showed a significant increase in Kmt2d mRNA compared to control cells. Apelin-13 treatment significantly suppressed H/R induced increase of Kmt2d mRNA in NRK-52E cells (Fig. 4E). In addition, significant increases in Ash and Kmt2d mRNA with no change in Set7/9 mRNA were found after H/R in the HBZY-1 cells, whereas apelin-13 treatment of HBZY-1 cells significantly suppressed the H/R-induced increase of Kmt2d mRNA, but not Ash mRNA (Fig. 4G).

Please cite this article as: H. Chen, et al., Apelin protects against acute renal injury by inhibiting TGF-β1, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbadis.2015.02.013

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Fig. 4. Apelin-13 suppresses I/R or H/R injury induced histone hypermethylation and in the kidneys, cultured renal cells. Representative Western blot analysis with quantitative densitometric results of H3K4me1, H3K4me2, H3K4me3, H3K9me3, H4K20me3, H3K79me1 and H3 are shown in (A). Representative PCR analysis with densitometric quantitative results of Ash1, Kmt2d, Set7/9, Set1, Dot1l, Kdm1a, Kdm5a and Rn18s were shown in (B). (C) Representative Kmt2d staining pictures for different experimental groups. Representative Western blot analysis with densitometric quantitative results of H3K4me2, H3K79me1 and H3 were shown in (E and G). Representative PCR analysis with densitometric quantitative results of Ash1, Kmt2d, Set7/9, and Rn18s were shown in (F and H). CT or NC, non-injured rats or cells; I/R or H/R, I/R injured rats or H/R injured cells; I/R or H/R + Ap, apelin-13 treated I/R injured rats or H/R injured cells, n = 3–7 per group. ⁎p b 0.05 compared to CT or NC group; #p b 0.05 compared to I/R or H/R group.

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To further investigate whether alteration of histone H3K4 methylation, per se, in the cultured renal cells can induce cell injury or not, TCP, an inhibitor of HMT [38], was used to treat NRK-52E cells. Compared to DMSO treated cells, TCP induced significant increases in H3K4 methylation in a dose-dependent manner (supplementary data, Fig S4). At the same time, the activation of caspase-8 and caspase-3, as well as the level of ICAM-1 were also dose-dependently induced by TCP treatment in NRK-52E cells (supplementary data, Fig. S4), indicating that apoptosis and inflammation might be induced by elevation of H3K4 methylation.

3.5. Apelin-13 inhibits I/R or H/R injury induced up-regulation of Tgf-β1

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TGF-β1 has long been implicated as a central mediator of tissue scarring, apoptosis and fibrosis in many disease models, including acute renal injury [45]. Significantly increased Tgf-β1 mRNA was evident in the kidneys after I/R injury, and apelin-13 treatment significantly reduced I/R-induced up-regulation of Tgf-β1 in the kidneys (Fig. 5A). A ChIP assay further demonstrated increased recruitment of H3K4me2 to the promoter of Tgf-β1, which was consistent with the increased

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Please cite this article as: H. Chen, et al., Apelin protects against acute renal injury by inhibiting TGF-β1, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbadis.2015.02.013

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Fig. 5. Apelin-13 inhibits I/R or H/R injury induced Tgf-β1 in kidneys and cultured renal cells. Representative PCR analysis with densitometric quantitative results of Tgf-β1 and Rn18s are shown in (A, C, D). (B) Quantitative analysis of PCR of chromatin immunoprecipitated DNA, which measures the binding affinity of H3K4me2 to the promoter of Tgf-β1. The abundance is relative to the input in the same sample with the same primer. CT or NC, non-injured rats or cells; I/R or H/R, I/R injured rats or H/R injured cells; I/R or H/R + Ap, apelin-13 treated I/R injured rats or H/R injured cells, n = 3 per group. ⁎p b 0.05 compared to NC group; #p b 0.05 compared to I/R or H/R group.

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3.6. Over-expression of apelin protects mesangial cells from H/R induced apoptosis and Tgf-β1 up-regulation

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In addition to direct apelin-13 treatment, the pPCAGSIH-apelin plasmid was used to study the effects of apelin over-expression in H/R injured mesangial cells. Apln mRNA was significantly decreased in H/R injured cells compared to control cells. Over-expression of apelin markedly up-regulated Apln mRNA level in HBZY-1 cells cultured under H/R conditions (Fig. 6A). No significant difference in APJ level was observed in HBZY-1 cells cultured under different conditions. Furthermore, overexpression of apelin significantly suppressed H/R-induced increased Hif1α level in HBZY-1 cells (Fig. 6B). Caspase-3 has been shown to be primarily responsible for the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1) during cell death [46]. Compared to control cells, elevated cell death in H/R stimulated cells was demonstrated by elevation of the cleaved, active form of caspase3, caspase-8 and PARP-1, and these changes were markedly attenuated by apelin over-expression (Fig. 6C).

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mRNA level of Tgf-β1 found in the kidneys of I/R injured rats. Apelin-13 treatment significantly inhibited the I/R-induced increase in H3K4me2 binding to the promoter of Tgf-β1 in the kidneys (Fig. 5B). Consistent with the in vivo study, significantly increased Tgf-β1 mRNA was also observed in tubular epithelial cells and mesangial cultured under H/R conditions, and apelin-13 treatment reduced H/R-induced elevation of Tgf-β1 (Fig. 5C and D).

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Fig. 6. Over-expression of apelin inhibits H/R activated apoptosis and Tgf-β1 in HBZY-1 cells. Representative PCR analysis with densitometric quantitative results of Apln, Rn18s were shown in (A). Representative Western blot analysis with densitometric quantitative results of APJ, Hif1α and β-actin were shown in (B). Representative Western blot analysis with densitometric quantitative results of caspase-3 (35 KD), c-cas3 (19 KD), c-cas3 (17 KD), c-cas8 (26KD), c-cas8 (18KD), PARP-1, c-PARP-1 and β-actin were shown in (C). Representative PCR analysis with densitometric quantitative results of Tgf-β1 and Rn18s are shown in (D). NC + β-gal, cells transfected with the pPCAGSIH-β-gal plasmid cultured under normal condition; H/R + β-gal, cells transfected with the pPCAGSIH-β-gal plasmid cultured under H/R condition; H/R + apelin, cells transfected with the pPCAGSIH-apelin plasmid cultured under H/R condition, n = 3 per group. *p b 0.05 compared to NC group; #p b 0.05 compared to H/R + β-gal group.

We next examined whether over-expression of apelin could suppress H/R up-regulated Tgf-β1 mRNA level in HBZY-1 cells. A significant increase of Tgf-β1 mRNA level in HBZY-1 cells after H/R injury was observed, which was markedly suppressed by over-expression of apelin (Fig. 6D).

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3.7. Over-expression of apelin alters histone methylation level in H/R 400 stimulated HBZY-1 cells 401 We also analyzed histone methylation in H/R injured HBZY-1 cells with apelin over-expression. Substantial increases in histone methylation (H3K4me2 and H3K79me1) was evident in HBZY-1 cells cultured under H/R conditions compared to controls, whereas this increase was

Please cite this article as: H. Chen, et al., Apelin protects against acute renal injury by inhibiting TGF-β1, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbadis.2015.02.013

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The number of AKI patients, as well as the population susceptible to AKI, such as diabetic and hypertension patients, is increasing worldwide, whereas an optimal therapy for AKI is still lacking [47,48]. Rodent renal I/R injury is a good model to study the pathogenesis of AKI and to seek possible therapies for the disease. A recent report suggested that administration of apelin-13 or leptin suppresses the renal I/R injury induced elevation of urea and pathological damage in rats [49], however the underlying mechanism for these beneficial effects remains unknown. In the present study, we report that apelin-13 significantly inhibits I/R injury induced renal dysfunction in rats. Furthermore, our results demonstrate that apelin-13 suppressed inflammation (ICAM-1 expression) and apoptosis (caspase-3 activation) in both renal I/R injured rats and renal cells cultured under H/R injury conditions. Thus, our data suggested that apelin-13 plays a protective role in renal I/R injury through its anti-apoptotic and anti-inflammatory action. Clinical studies have demonstrated that the level of apelin is significantly reduced in kidney allograft recipients with coronary artery disease and the patients with autosomal dominant polycystic kidney disease [50]. Consistent with the clinical observations, in the rodent hypertension model induced by two-kidney-one-clip (2K1C), reduced serum level of apelin but not Ang II and arginine-vasopressin is also observed, accompanied by significantly decreased mRNA and protein levels of APJ in ischemic kidneys [51]. However, administration of apelin-13 (20 μg/kg) significantly decreased systolic and diastolic blood pressures in these 2K1C-induced hypertensive rats [51]. Furthermore, apelin-13 showed significant protective effects on diabetic nephropathy in two different type 1 diabetic mouse models [24,52].

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Fig. 8. Possible mechanisms behind the beneficial effects of apelin on renal I/R injury.

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4. Discussion

Taken together, including the present findings, the apelin is a new biomarker whose level can predict renal function and apelin-13 appears to be a good therapeutic candidate for chronic and acute renal diseases, at least in rodent models. TGF-β1 has been firmly established as a central mediator of kidney fibrosis and inflammation associated with progressive kidney diseases [6,7]. During H/R injury, hypoxia caused by vascular insufficiency may enhance TGF-β1 synthesis [53,54]. Recent studies have suggested that the crucial role of TGF-β1 in collagen synthesis and fibrosis may be antagonized and/or reduced by the action of certain agents, such as proteoglycan decorin or KS370G [55,56], which inhibit TGF-β1 level and exert a protective effect of kidneys in renal I/R injury [56,57]. In the present study, administrating apelin-13 or over-expressing apelin both significantly suppressed I/R and H/R induced elevated Tgf-β1 in kidneys and cultured renal cells. Furthermore, apelin-13 treatment also inhibits the I/R and H/R induced elevated Tgf-β1 in the kidneys and cultured renal cells. Thus, the anti-apoptotic and anti-inflammatory actions of apelin13 on renal I/R injury are due to its inhibitory role on Tgf-β1. In the present study, among the altered histone methylation and HMTs levels, the elevation of H3K4me2 and upregulation of Kmt2d was consistently found in I/R injured kidneys and cultured renal cells, and these I/R induced alterations were markedly inhibited by apelin13. KMT2D mutations have been found in the patients with clear-cell renal cell carcinoma or Kabuki syndrome [58]. Furthermore, KMT2D mutations in Kabuki syndrome patients are more likely to have kidney anomalies than those patients without KMT2D mutation [59,60], suggesting dysfuncation or abnormal level of KMT2D may contribute to the development of renal diseases. In the present study, we also demonstrated that after renal I/R injury, elevated H3K4me2 bound to the promoter of Tgf-β1 which lead to increased transcription of Tgf-β1, and apelin-13 treatment down-regulated Tgf-β1 as well as reduced H3K4me2 bound to the promoter of Tgf-β1 after renal I/R injury. Further studies are required to determine whether Kmt2d can directly bind to the promoter of Tgf-β1 and regulate transcription of Tgf-β1. Through regulation of histone modifications and the responsible enzymes, apelin-13 may transform the action of a short-term elevation of this adipocytokine into a long-lasting signaling pathway change. However, the critical role of Kmt2d in renal I/R injury and whether it is the major target for apelin-induced renoprotection in renal I/R injury awaits further investigation. In summary, this study revealed a novel mechanism underlying the anti-inflammatory, anti-apoptotic and anti-fibrotic action of apelin-13, which confers protection against I/R injury in the kidney by epigenetic regulation. Either exogenous apelin-13 treatment or endogenous expression Apln in cultured renal cells also demonstrated the cell death

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significantly abrogated by apelin over-expression (Fig. 7A, B). We next examined the mRNA levels of Ash1, Kmt2d in HBZY-1 cells cultured under normal or H/R conditions. Only the mRNA level of Kmt2d was significantly increased in HBZY-1 cells cultured under H/R condition compared with control cells, but was markedly reduced in the H/R treated cells that over-express apelin (Fig. 7C, D).

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Fig. 7. Over-expression of apelin regulates H/R induced histone methylation in HBZY-1 cells. Representative western blot analysis of H3K4me2, H3K79me1 and H3 were shown in (A), with densitometric quantitative results in (B). Representative PCR analysis of Ash1, Kmt2d and Rn18s were shown in (C), with densitometric quantitative results in (D). NC + β-gal, cells transfected with the pPCAGSIH-β-gal plasmid cultured under normal condition; H/R + β-gal, cells transfected with the pPCAGSIH-β-gal plasmid cultured under H/R condition; H/R + apelin, cells transfected with the pPCAGSIH-apelin plasmid cultured under H/R condition, n = 3 per group. *p b 0.05 compared to NC group; #p b 0.05 compared to H/R + β-gal group.

Please cite this article as: H. Chen, et al., Apelin protects against acute renal injury by inhibiting TGF-β1, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbadis.2015.02.013

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protective role of apelin-13 on reducing Tgf-β1 after in vitro H/R injury (Fig. 8). All these suggest that apelin-13 may be considered as a viable strategy to prevent acute cell damage and improve the outcome of I/R injury to the kidneys.

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Competing interests The authors declare no competing interest. Transparency Document

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The Transparency document associated with this article can be found, in the online version

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Acknowledgements

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We thank Dr. Takakura (Osaka University) for apelin plasmid. This work was supported by the National Basic Research Program of China (2012CB524901), the Natural Science Foundation of China (Nos. 81202557, 31271370, 81172971, 81222043 and 31471208), Central University Basic Research Funds (to L.Z.), the Municipal Key Technology Program of Wuhan (Wuhan Bureau of Science & Technology, No. 201260523174), the Health Bureau of Wuhan (WX12B06) and the Natural Science Foundation of Hubei Province (2013CFB359 and 2014CF021).

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Appendix A. Supplementary data

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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbadis.2015.02.013.

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References

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Please cite this article as: H. Chen, et al., Apelin protects against acute renal injury by inhibiting TGF-β1, Biochim. Biophys. Acta (2015), http:// dx.doi.org/10.1016/j.bbadis.2015.02.013

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