The Histone Methyltransferase Enzyme Enhancer of Zeste Homolog 2 Protects Against Podocyte Oxidative Stress and Renal Injury in Diabetes

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The Histone Methyltransferase Enzyme Enhancer of Zeste Homolog 2 Protects against Podocyte Oxidative Stress and Renal Injury in Diabetes

† Ferhan S. Siddiqi,* Syamantak Majumder,* Kerri Thai,* Moustafa Abdalla,* Pingzhao Hu, ‡ Suzanne L. Advani,* Kathryn E. White, Bridgit B. Bowskill,* Giuliana Guarna,* Claudia C. dos Santos,* Kim A. Connelly,* and Andrew Advani*

*Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute, St. Michael’s Hospital, Toronto, Ontario, Canada; †Department of Biochemistry and Medical Genetics and George and Fay Yee Centre for Healthcare Innovation, University of Manitoba, Winnipeg, Manitoba, Canada; and ‡Electron Microscopy Research Services, Newcastle University, Newcastle upon Tyne, United Kingdom

ABSTRACT Epigenetic regulation of oxidative stress is emerging as a critical mediator of diabetic nephropathy. In diabetes, oxidative damage occurs when there is an imbalance between reactive oxygen species generation and enzymatic antioxidant repair. Here, we investigated the function of the histone methyltransferase enzyme enhancer of zeste homolog 2 (EZH2) in attenuating oxidative injury in podocytes, focusing on its regulation of the endogenous antioxidant inhibitor thioredoxin interacting protein (TxnIP). Pharmacologic or genetic depletion of EZH2 augmented TxnIP expression and oxidative stress in podocytes cultured under high-glucose conditions. Conversely, EZH2 upregulation through inhibition of its regulatory microRNA, microRNA-101, downregulated TxnIP and attenuated oxidative stress. In diabetic rats, depletion of EZH2 decreased histone 3 lysine 27 trimethylation (H3K27me3), increased glomerular TxnIP expression, induced podocyte injury, and augmented oxidative stress and proteinuria. Chromatin immunoprecipitation sequencing revealed H3K27me3 enrichment at the promoter of the transcription factor Pax6, which was upregulated on EZH2 depletion and bound to the TxnIP promoter, controlling expression of its gene product. In high glucose–exposed podocytes and the kidneys of diabetic rats, the lower EZH2 expression detected coincided with upregulation of Pax6 and TxnIP. Finally, in a gene expression array, TxnIP was among seven of 30,854 genes upregulated by high glucose, EZH2 depletion, and the combination thereof. Thus, EZH2 represses the transcription factor Pax6, which controls expression of the antioxidant inhibitor TxnIP, and in diabetes, downregulation of EZH2 promotes oxidative stress. These ﬁndings expand the extent to which epigenetic processes affect the diabetic kidney to include antioxidant repair.

J Am Soc Nephrol 27: 2021–2034, 2016. doi: 10.1681/ASN.2014090898

Although the long–term follow-up of individuals as a result of an imbalance between reactive oxygen enrolled in trials of glycemic lowering has drawn species (ROS) generation through increased attention to the potential role that epigenetic processes may play in the development of diabetes complications,1,2 it is evident that the effects of Received September 17, 2014. Accepted September 22, 2015. these processes extend beyond their role in meta- F.S.S. and S.M. contributed equally to this work. 3 bolic memory. Ever since the importance of epi- Published online ahead of print. Publication date available at genetic mechanisms in mediating glucose–induced www.jasn.org. ﬁ cellular injury was rst realized, a link has been Correspondence: Dr. Andrew Advani, St. Michael’sHospital, drawn between chromatin modiﬁcations and oxi- 6-151, 61 Queen Street East, Toronto, ON, Canada M5C 2T2. dative damage.4 Oxidative stress is a major contrib- Email: [email protected] utor to cellular dysfunction in diabetes, occurring Copyright © 2016 by the American Society of Nephrology

J Am Soc Nephrol 27: 2021–2034, 2016 ISSN : 1046-6673/2707-2021 2021 BASIC RESEARCH www.jasn.org mitochondrial flux on the one hand and impaired enzymatic preincubation with DZNep, high glucose (25 mM), or a com- antioxidant repair mechanisms on the other hand.5 Recently, bination of high glucose and DZNep. Exposure of cultured glucose-induced augmentation of mitochondrial ROS podocytes to high glucose resulted in a significant generation production was shown to be under epigenetic regulation.6 of ROS over 12 hours compared with cells incubated in either However, whether antioxidant repair mechanisms may also normal glucose (5.6 mM) or mannitol (osmotic control) (Figure be regulated by epigenetic processes has yet to be scrutinized. 1C). Treatment with DZNep in the presence of high glucose The histone methyltransferase enzyme enhancer of zeste significantly augmented ROS to levels greater than high glucose homolog 2 (EZH2) is the catalytic subunit of the multimeric alone (Figure 1D). To evaluate whether programmed cell death Polycomb Repressive Complex 2 (together with Suz12, Eed, was detectable in podocytes exposed to high glucose and/or and RbAP46/48), which ordinarily trimethylates lysine residue DZNep, we performed an assessment of terminal deoxynucleotidyl 27 on histone 3 (H3K27me3), essentially repressing gene transferase–mediated dUTP nick–end labeling (TUNEL) –positive expression by strategic modification of chromatin structure. nuclei. Mimicking the effects on ROS, the combination of EZH2 regulates pancreatic b–cell regeneration7 and adipogen- high glucose and DZNep increased podocyte cell death, esis,8 and bioinformatic strategies have found that depletion of whereas either high glucose or DZNep alone was without ef- the histone methyltransferase can increase oxidative damage fect (Figure 1E). in cancer cells9 through augmented expression of the endog- Having established that, in the presence of high-glucose enous antioxidant inhibitor, thioredoxin interacting protein concentrations, EZH2 depletion with DZNep induces oxida- (TxnIP).10 tive stress and podocyte cell death, we next sought to determine Possessing a carbohydrate response element within its whether the histone methyltransferase regulates expression of promoter region, TxnIP is a glucose-responsive gene and crit- the antioxidant inhibitor TxnIP in podocytes. Using quanti- ical regulator of the cellular redox state,11 where it promotes tative RT-PCR, we found that DZNep and high glucose oxidative injury primarily by antagonizing the actions of the independently increased TxnIP transcript abundance and endogenous antioxidant, thioredoxin (Trx).12 In our earlier that, in the setting of DZNep treatment, high-glucose con- work, we found TxnIP to be upregulated in renal cells exposed centrations promoted a further increase in TxnIP mRNA to high-glucose concentrations, kidneys of diabetic rodents, compared with either condition in isolation (Figure 1F). As and biopsies obtained from patients with diabetic nephropa- expected, a parallel set of experiments to evaluate TxnIP pro- thy, where it functioned to promote ROS accumulation and tein abundance by Western blot revealed a similar pattern of matrix production.5 change with augmented TxnIP expression observed by the In this study, we sought to examine whether EZH2 regulates combination of high glucose and DZNep (Figure 1G). To de- TxnIPexpression, oxidative stress, and renal injury in diabetes. terminewhethertheadditiveeffectsofDZNepandhigh In light of emerging evidence that epigenetic mechanisms play glucose on TxnIP expression are cell type dependent, we mea- homeostatic roles in terminally differentiated cells,13 especially sured mRNA levels in cultured rat mesangial cells, proximal under diabetic conditions,3,6,14 we focused our experiments tubule lineage NRK-52E cells, and MDCK cells, which are of a on the effects of this pathway in podocytes, the final barriers to distal tubule/collecting duct lineage.5 Illustrating the cell- protein leakage into the urinary filtrate, in which early injury specific effects of EZH2 inhibition with DZNep, we found may contribute to the subsequent development of diabetic that, whereas both DZNep and high glucose independently glomerulosclerosis.15 increased TxnIP expression in each cell line, an additive effect (as observed in podocytes) was seen in mesangial cells but not in NRK-52E cells or MDCK cells (Supplemental Figure 1). To RESULTS evaluate the critical role of TxnIP in podocyte oxidative damage, we transfected cells with either TxnIP short–hairpin RNA EZH2 Depletion Upregulates TxnIP and Promotes (shRNA) or a vector containing a constitutively active Trx gene Oxidative Stress and Programmed Cell Death in (Supplemental Figure 2). In these experiments, either TxnIP Podocytes knockdown or Trx overexpression abrogated the increase in To examine the effects of constitutive EZH2 in podocytes, we ROS levels induced by the combination of high glucose and took advantage of the S-adenosylhomocysteine hydrolase in- DZNep (Figure 1H). hibitor 3-deazaneplanocin A (DZNep) that is known to induce EZH2 degradation.16 Initial immunoblotting experiments TxnIP Expression and Oxidative Stress Are Increased confirmed the presence of EZH2 protein in cultured mouse by EZH2 Knockdown and Decreased by EZH2 podocytes and its depletion with DZNep (Figure 1A) together Upregulation through MicroRNA-101 Inhibition with a concurrent reduction in H3K27me3 levels (Figure 1B). Cognizant that, in cancer cells, DZNep has been reported to To determine whether EZH2 depletion affects the generation alter the methylation status of some other histone lysine of oxidative stress in podocytes, we measured intracellu- residues in addition to H3K27me3,17 we immunoblotted ly- lar ROS levels using the fluorescent oxidation product of sates of DZNep-treated podocytes for a number of other 29,79-dichlorodihydrofluorescein diacetate (CFDA) after histone marks (specifically H3K4me3, H3K9me2, and

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H3K79me3). Whereas H3K4me3 and H3K79me3 were unaffected, H3K9me2 levels were reduced by DZNep along with the expected decrease in H3K27me3 (Figure 1B, Supplemental Figure 3). Because H3K9 dimethylation is primarily regulated by the histone methyltransferase G9a rather than EZH2,18 to confirm that the effect of DZNep on podocyte oxidative stress was ostensibly caused by EZH2 inhibition, knockdown experiments were performed by transfecting podocytes with a sequence–specific EZH2 shRNA (Supple- mental Figure 4). Consistent with results obtained with the small molecule, in the presence of high glucose, EZH2 shRNA increased TxnIP mRNA (Figure 2A), augmented ROS accumulation (Figure 2B), and increased podocyte cell death (Figure 2C). Having discovered that either DZNep treatment or EZH2 knockdown augmented TxnIP expression and oxidative stress in podocytes, we next asked the question as to whether upregulation of the histone methyltransferase would attenuate these processes. We, thus, carried out a series of experiments targeting an established microRNA (miR) regulator of EZH2, miR-101.19 To determine whether miR-101 inhibition may augment EZH2 expression, we used a sequence–specific oligonucleotide inhibitor to antagonize its effects in podocytes in vitro.Treatment with this miR-101 inhibitor resulted in a Figure 1. EZH2 depletion with DZNep augments podocyte TxnIP expression and oxi- striking upregulation of podocyte EZH2 dative stress. Effect of DZNep or high glucose on EZH2 and TxnIP expression, ROS levels, expression determined by immunoblotting and programmed cell death in cultured mouse podocytes. (A and B) Immunoblotting (Figure 2D). Upregulation of EZH2, in- mouse podocytes for (A) EZH2 and (B) H3K27me3 under control conditions or after DZNep duced by miR-101 inhibition, was accom- treatment for 48 hours. (C) CFDA fluorescence intensity in mouse podocytes exposed to panied by decreased podocyte TxnIP control (5.6 mM glucose), high glucose (25 mM) for 1, 3, or 12 hours, or mannitol (osmotic control). (D) CFDA fluorescence intensity in mouse podocytes under control conditions or expression (Figure 2E) and attenuation of treated with DZNep, high glucose, high glucose and DZNep, or mannitol. All values are ROS generation (Figure 2F) in podocytes normalized to control. (E) Quantitation of TUNEL-positive nuclei (%) from cultured mouse cultured under high-glucose conditions, podocytes incubated under conditions of normal glucose, DZNep, high glucose, high also reducing ROS levels to below the glucose and DZNep, or mannitol. (F and G) Change in TxnIP expression by (F) real-time baseline of untreated cells. PCR for TxnIP mRNA and (G) Western blot for TxnIP protein in cultured mouse podocytes incubated for 48 hours under the following conditions: control, DZNep, high glucose, a combination of high glucose and DZNep, or mannitol. (H) CFDA fluorescence intensity in †† podocytes transfected with scrambled shRNA, TxnIP shRNA, or a constitutively active between high glucose and DZNep; P,0.001 ‡‡ vector overexpressing Trx and incubated with either normal glucose or a combination of versus control or DZNep; P,0.001 versus all high glucose (12 hours) and DZNep for 48 hours. All values are normalized to control or other groups except high glucose (P,0.05); || scrambled as indicated. AU, arbitrary unit; HG, high glucose. *P,0.05 versus control; §§P,0.001 versus scrambled; P,0.001 ver- † ‡ P,0.01 versus control; P,0.05 versus control or DZNep; §P,0.001 versus all other sus high glucose and DZNep and P,0.05 | groups except high glucose (P,0.01); P,0.001 versus control by two-way ANOVA; versus mannitol; ¶¶P,0.001 versus high glu- ¶P,0.001 versus all other groups except mannitol (P,0.001); **P,0.05 for an interaction cose and DZNep.

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Figure 2. EZH2 knockdown augments TxnIP expression and EZH2 augmentation through miR-101 inhibition attenuates TxnIP expression. Effect of EZH2 knockdown or upregulation through miR-101 inhibition on podocyte TxnIP expression and oxidative stress. (A–C) Effect of shRNA–mediated EZH2 knockdown. (A) TxnIP gene expression by real-time PCR, (B) CFDA ﬂuorescence intensity, and (C) quantitation of TUNEL-positive nuclei (%) in mouse podocytes transfected with scrambled shRNA, incubated with 25 mM (high) glucose (control), or transfected with EZH2 shRNA in the presence of high glucose. (D–F) Effect of miR-101 inhibition. (D) Western blot for EZH2 expression in mouse podocytes treated with vehicle (control) or miR-101 inhibitor for 48 hours. (E) TxnIP protein expression by Western blot and (F) CFDA ﬂuorescence intensity in podocytes under the following conditions: normal glucose, miR-101 inhibitor, high glucose for 12 hours, and high glucose (12 hours) in the presence of miR-101 inhibitor. All values are normalized to control except † TUNEL. AU, arbitrary unit; HG, high glucose. *P,0.01 versus scrambled; P,0.001 versus scrambled or high glucose (control); ‡ | P,0.001 versus scrambled or high glucose (control); §P,0.01 versus control; P,0.001 versus high glucose; ¶P,0.05 versus control; †† **P,0.001 versus high glucose; P,0.01 versus high glucose.

DZNep Treatment of Diabetic Rats Promotes Podocyte period, because we had previously found renal TxnIP expres- Injury, Oxidative Stress, and Proteinuria sion to already be increased in diabetic rats after 3 weeks (with- Having unearthed a role for the histone methyltransferase out further increase on longer follow-up)5 and because we EZH2 in repressing podocyte TxnIP expression and oxidative surmised that this timeframe would provide the opportunity stress in vitro, we set out to determine whether the same mech- to probe for an anticipated augmentation of renal injury ex- anism is also active in vivo. Initial immunoblotting experi- pected from our in vitro observations. Animal characteristics ments of kidney homogenates conﬁrmed that treatment of are shown in Table 1. Without affecting blood glucose or BP, rats by twice weekly intraperitoneal (ip) injection of DZNep DZNep treatment signiﬁcantly increased urine protein excre- caused a reduction in H3K27 trimethylation (Figure 3A). Sub- tion in diabetic rats, whereas there was no change in normo- sequently, we randomly allocated control and streptozotocin glycemic animals (Figure 3B). (STZ) diabetic rats to receive either DZNep or vehicle by twice Toevaluate the effects of DZNep on renal structure, we used weekly ip injection for 3 weeks. We selected this treatment transmission electron microscopy to carefully examine the

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Figure 3. DZNep treatment augments proteinuria, podocytopathy, glomerular TxnIP expression, and oxidativestressindiabetic rats. (A) Western blot analysis of H3K27me3 and total histone H3 expression in rat kidney homogenates in control and DZNep- treated animals after 5 days (n=4 per group). (B) Measurement of urinary protein excretion in control and diabetic rats treated with vehicle or DZNep for 3 weeks (n=12 per group). (C–F) Representative podocyte ultrastructure by transmission electron microscopy from (C and D) control and (E and F) diabetic rats treated with (C and E) vehicle or (D and F) DZNep. The asterisks in D and F mark the presence of (D) adsorption droplets or (F) vacuoles in podocytes from (D) a control rat treated with DZNep and (F) a diabetic rat treated with DZNep. (G–J) Transmission electron micrographs of podocyte foot processes from (G and H) control and (I and J) diabetic rats treated with (G and I) vehicle or (H and J) DZNep. The arrows in I and J mark areas of foot process effacement in diabetic rats treated with (I) vehicle or (J) DZNep. (K) Quantitation of podocyte abnormalities (%). (L–O) Glomerular TxnIP immunostaining of rat kidney sections in (L and M) control and (N and O) diabetic rats treated with (L and N) vehicle or (M and O) DZNep for 3 weeks. Original magniﬁcation, 3400. (P) Quantitation of glomerular TxnIP immunostaining. (Q) Urinary 8-hydroxy-29- † deoxyguanosine (8-OHdG) excretion. AU, arbitrary unit. *P,0.05 versus control; P,0.001 versus control and vehicle, P,0.001 ‡ versus control and DZNep, and P,0.05 versus diabetes; P,0.05 versus all other groups; §P,0.001 versus control and vehicle, | P,0.05 versus control and DZNep, and P,0.05 versus diabetes; P,0.001 versus control and vehicle or control and DZNep; ¶P,0.01 versus control and vehicle or control and DZNep; **P,0.01 versus diabetes and vehicle.

glomerular ultrastructure of both control and diabetic rat staining were each unaffected by DZNep treatment in diabetic kidneys with and without DZNep treatment. Consistent with rats (Supplemental Figure 5). our in vitro data, diabetic rats treated with DZNep had ultra- We next sought to determine whether the podocyte injury structural evidence of increased podocyte abnormalities, in- observed in diabetic rats treated with DZNep was accompanied cluding vacuolization and foot process effacement (Figure 3, by enhanced TxnIP expression and oxidative stress. Consistent C–K). By way of contrast, glomerular capillary density, with our observations in cultured cells, immunostaining of mesangial matrix deposition, and tubule epithelial TUNEL kidney sections revealed heightened glomerular TxnIP in the

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Table 1. Animal characteristics of control and diabetic Sprague–Dawley rats treated with vehicle or DZNep for 3 weeks Body Kidney Kidney Weight:Body Blood Glucose Hemoglobin Hemoglobin Systolic Animal Groups Weight (g) Weight (g) Weight (%) (mmol/L) A1c (%) A1c (mmol/mol) BP (mmHg) Control + vehicle 519611 1.5660.05 0.30060.005 6.660.2 5.060.1 3261 12066 Control + DZNep 496613 1.6160.05 0.32560.009 6.560.2 4.860.1 2961 12765 Diabetes + vehicle 344611a,b 1.6060.05 0.46960.016a,b 28.960.8a,b 12.560.2a,b 11362a,b 13067 Diabetes + DZNep 31468a,b 1.5060.04 0.47960.011a,b 27.861.0a,b 12.260.2a,b 11163a,b 13667 Data are presented as means6SEM. aP,0.001 versus control and vehicle. bP,0.001 versus control and DZNep. kidneys of diabetic rats treated with DZNep (Figure 3, L–P). EZH2 to the regulation of TxnIP, we observed that Pax6 itself Similarly, urinary 8-hydroxy-29-deoxyguanosine, a biomarker binds to the TxnIP promoter (Figure 4F) and that Pax6 knock- of oxidative stress, was elevated in diabetic rats and further down with short interference RNA (Supplemental Figure 10) augmented by DZNep treatment in these animals (Figure 3Q). prevented the upregulation of TxnIP by DZNep treatment Finally, to discern whether augmentation of renal injury (Figure 4G). Collectively, these results define a homeostatic with DZNep is pervasive across kidney diseases, we adminis- role for EZH2 in repressing the transcription factor Pax6 and tered the inhibitor to mice 24 hours before induction of hence, the antioxidant inhibitor, TxnIP,in podocytes. Support- ischemia-reperfusion injury (IRI) by bilateral renal clamping ing dysregulation of this process in diabetes, we found that for 30 minutes. Supporting a disease-specific effect of EZH2 EZH2 expression was reduced, coinciding with upregulation inhibition, pretreatment with DZNep attenuated both the rise of both Pax6 and TxnIP in glomeruli isolated from STZ dia- in plasma creatinine and BUN and the tubular injury in mice betic rats (Figure 4, H–K). Similarly, exposure of cultured po- 24 hours after IRI (Supplemental Figure 6). docytes to high-glucose concentrations resulted in a decrease in EZH2 mRNA abundance and increase in Pax6 (Supplemental EZH2 Regulates the Transcription Factor Paired Box 6, Figure 11) and TxnIP (Figure 1F). which Itself Controls TxnIP Expression Because our experiments had unearthed a role for EZH2 in EZH2 Regulates Multiple Biologic Processes in attenuating expression of the antioxidant inhibitor, TxnIP, Podocytes and hence, oxidative stress in podocytes, we next turned our Finally, although we had focused our experiments on the attention to the mechanism by which EZH2 represses TxnIP regulation of TxnIP by EZH2 and its role in oxidative stress, we expression in podocytes. Supporting the repression of TxnIP were cognizant that the function of the histone methyltrans- promoter activity by EZH2, a reporter assay showed that ferase in regulating cellular behavior extends beyond repres- treatment of podocytes with DZNep resulted in a 50% increase sion of the endogenous antioxidant inhibitor. To explore the in luciferase activity (Figure 4A). To determine whether EZH2 biologic processes under the influence of EZH2 in podocytes, represses TxnIP by trimethylating H3K27 at its promoter region, we we performed gene expression analyses using an Illumina performed chromatin immunoprecipitation (ChIP) –sequencing Mouse WG-6 v2.0 Expression BeadChip, which independently experiments after immunoprecipitation of DNA with an confirmed upregulation of TxnIP with DZNep treatment (fold H3K27me3-specific antibody. Interestingly, in this analysis, change of 1.25; P=0.002). From a panel of 30,854 genes, the we did not observe an enrichment of H3K27me3 at the TxnIP expression of 18 genes was altered by DZNep, high glucose, gene of mouse podocytes (Supplemental Figure 7). This obser- and the combination of DZNep and high glucose, with TxnIP vation led us to reason that EZH2 may regulate TxnIP expres- being one of seven to be upregulated (Figure 5). Bioinformatic sion through the repression of trans-acting factor(s). In a comparison of control and DZNep-treated cells revealed five search for what these factor(s) may be, we performed an in biologic processes to be significantly enriched in podocytes silico screen of possible transcription factor binding sites within with EZH2 depletion, four of which included the presence the TxnIP promoter (Supplemental Figure 8, Supplemental of TxnIP (protein localization, protein transport, establish- Table 1). Of 71 possible candidates, the transcription factor ment of protein localization, and cell cycle) (Supplemental paired box 6 (Pax6) was unique in being enriched for Figure 12). Supplemental Tables 3–5 show biologic process, H3K27me3 at its promoter in podocytes compared with either molecular function, and cellular compartment enrichment in input DNA or podocytes treated with DZNep (Figure 4B, Sup- DZNep-treated podocytes. plemental Figure 9, Supplemental Table 2). Separate ChIP experiments confirmed an approximately 100-fold enrichment of H3K27me3 at the Pax6 promoter (Figure 4C) and an DISCUSSION increase in Pax6 expression in podocytes after DZNep treatment (Figure 4, D and E). In additional experiments The post-translational modification of histone proteins by supporting a critical role for Pax6 as an intermediate linking the addition or removal of functional groups can have

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Figure 4. The transcription factor Pax6 mediates the regulation of TxnIP expression by EZH2 in podocytes. (A) Luciferase promoter reporter assay of podocytes incubated under control conditions or after treatment with DZNep for 48 hours. (B) ChIP sequencing of the Pax6 promoter and gene regions after enrichment by immunoprecipitation with an anti-H3K27me3 antibody. Increased H3K27me3 at the Pax6 promoter is shown in control podocytes compared with either input DNA or podocytes treated with DZNep for 48 hours. (C) ChIP of the Pax6 promoter after H3K27me3 enrichment. (D and E) Increased Pax6 (D) mRNA and (E) protein after treatment with DZNep for 48 hours. (F) ChIP of the TxnIP promoter after Pax6 enrichment. (G) Increased TxnIP mRNA in podocytes after treatment with DZNep for 48 hours and prevention of TxnIP upregulation by transfection of cells with siRNA directed against Pax6. (H–K) Immunoblotting glomeruli isolated from control rats (n=4) or rats after 3 weeks of STZ-induced diabetes (n=7) for (I) EZH2, (J) Pax6, and (K) TxnIP. AU, † ‡ arbitrary unit; IP, immunoprecipitation. *P,0.01 versus control; P,0.05 versus IgG; P,0.001 versus IgG; §P,0.01 versus all other conditions; ¶P,0.05 versus control.

profound effects on the expression of genes in close prox- limit oxidative damage and renal dysfunction and is dys- imity. Lysine residue 27 on histone H3, for instance, may be regulated in diabetes. modified by the addition of one, two, or three methyl groups Although the hypothesis that altered chromatin modifica- by specific histone methyltransferase enzymes, where trime- tions may underlie the cellular memory for glucose was first thylation of H3K27 by EZH2 at the promoter region of target proposed almost one quarter of a century ago,20 it is only more genes may lead to transcriptional silencing. By combining recently that advancement in pharmacologic and genetic tools ChIP sequencing and in silico analyses with experiments has enabled epigenetic processes to be viewed as legitimate conducted in cultured cells and diabetic rodents, we report therapeutic targets. Most advanced in this regard is the addi- the transcriptional repression of the transcription factor tion or removal of functional acetyl groups on histone pro- Pax6 and consequently, the glucose–responsive gene TxnIP teins, where pharmacologic inhibition of deacetylase enzymes by EZH2 in podocytes. This pathway effectively functions to attenuates renal growth, albuminuria, and mesangial matrix

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through the development of DZNep, which was ﬁrst synthesized in 198624 and previously studied for its antiviral properties,25 but more recently, it was found to deplete cells of EZH2 through increased protein degradation, decreasing H3K27 trimethylation.16 To explore the role of EZH2 in glucose– induced cellular injury, we focused our attention on the regulation of TxnIP, an endogenous antioxidant inhibitor. In diabetes, ROS production commonly results from increased intracellular glucose metab- olism and subsequent donation of excess electrons to the mitochondrial electron transport chain that, after exceeding a homeostatic voltage threshold, leads to the generation of oxygen free radicals.26 Topro- tect against the deleterious effects of accumulating oxidative damage, cells possess a number of enzymatic antioxidant defense mechanisms, among them Trx, which ordinarily repairs oxidative damage of sulphydryl groups on cysteine and me- thionine residues.27 Trx itself is negatively regulated by the endogenous inhibitor TxnIP, which binds the enzyme inhibiting its bioavailability. Although both molecu- lar28 and topological5 observations indicate that the role of TxnIP in promoting oxidative injury extends beyond its negative regulation of Trx, augmented expression of the protein has been repeatedly associated with the development of diabetes complications.5,29–32 In this study, we found that depletion of Figure 5. TxnIP upregulation in a gene expression array of podocytes exposed to EZH2, with either DZNep or shRNA, DZNep and high glucose (HG). Gene expression analysis of podocytes after exposure rendered cultured podocytes vulnerable to DZNep, high glucose or HG and DZNep for 48 hours compared with control to the deleterious effects of high-glucose conditions. (A) Venn diagram of pairwise differential comparisons: 18 genes are dif- concentrations, leading to an increase in ferentially expressed (adjusted P value ,0.05) between control and the three other ROS levels and cell death. The augmenta- conditions. (B) Using the 18-gene intersection, the individual arrays were clustered. – DZNep- and HG- and DZNep-treated podocytes are observed to cluster together; HG tion of high glucose induced oxidative clusters closer to these groups than it does to control. TxnIP is one of seven genes to damage in podocytes was, at least in part, be upregulated in the three treatment states. mediated by derepression of TxnIP shown by (1) increased podocyte TxnIP mRNA and protein abundance when EZH2- deposition in diabetic rodents.21,22 Unlike acetylation, histone depleted cells were exposed to high-glucose concentrations methylation changes are often sustained down the cellular and (2) abrogation of ROS accumulation by either TxnIP lineage and thus, have received relatively greater attention knockdown or Trxoverexpression. In vivo, we observed a similar for their putative role in glycemic memory.4,23 By contrast, response, whereby treatment of diabetic rats with DZNep in- studies examining the cellular consequences of pharmacolog- duced podocyte injury and proteinuria accompanied by up- ically modifying these marks have been relatively sparse. In- regulation in glomerular TxnIP and biomarker evidence of deed, to our knowledge, this report represents the ﬁrst study augmented oxidative stress, albeit that these experiments examining the effects of in vivo histone methyltransferase in- were limited by the relatively short duration of study. Inter- hibition in diabetes complications. This opportunity arose estingly, however, even without DZNep treatment, EZH2

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was reduced in the glomeruli of STZ diabetic rats after 3 weeks. This observation together with the incremental increase in proteinuria and podocyte injury with DZNep are suggestive of slow basal H3K27me3 turnover that is accelerated by EZH2 inhibition.33 The mechanisms by which EZH2 facil- itates transcriptional repression involve the recruitment of enzymes that post- translationally modify histone proteins at other sites promoting chromatin compac- tion34 and DNA methyltransferases that methylatecytosineresidueswithinCpGis- lands at the promoter region of target genes.35 Histone modiﬁcation and DNA methylation represent the two major mechanisms by which a cell may epigenetically control gene expression, and both enjoy a bidirectional regulatory relation- ship with noncoding RNAs.36 miRs are short, noncoding RNAs that exert their silencing effects by binding to their target RNAs and preventing translation or promoting degradation.36 EZH2 is under the regulatory control of several miRs, including but not limited to miR-26a, miR-98, miR-101, miR-124, and miR-138.9,37,38 The best characterized of these is miR-101.39 Consistent with the effect of miR-101 in inhibiting EZH2 translation, we observed a profound increase in EZH2 protein in podocytes exposed to an oligonucleotide inhibitor of this miR. More- over, enhanced EZH2 translation mediated through inhibition of miR-101 was accompanied by an attenuation in both TxnIP expression and ROS accumulation. Although DZNep has been suggested as having broader effects on histone methylation than solely those at the H3K27 site,17 a piv- otal role for EZH2 in regulating oxidative stress is supported by both replication of the major ﬁndings with shRNA and repression of ROS accumulation through EZH2 augmentation with miR-101 inhibition.

(red) decreases H3K27me3 at the Pax6 promoter and increases Pax6 expression. Pax6, in turn, binds to the TxnIP promoter, augmenting TxnIP expression in podocytes, which in the setting of high-glucose concentrations, im- Figure 6. EZH2 regulates podocyte oxidative stress and renal injury in diabetes. The pairs antioxidant repair, leading to podocyte histone methyltransferase EZH2 is the catalytic subunit of Polycomb Repressive injury andrenaldysfunction. ThemiR, miR-101, Complex 2 and trimethylates H3K27 at the promoter region of the Pax6 gene re- limits EZH2 translation, and high glucose pressing expression of the transcription factor (blue). Depletion of EZH2 with DZNep downregulates EZH2.

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Whereas enrichment of H3K27me3 at the TxnIP promoter translation, transportation, and localization of proteins. may occur in certain cancer cells,10 our ChIP-sequencing ex- Thus, although the study of epigenetic mechanisms in the periments and subsequent validations studies determined adult kidney and diabetes complications is still in its relative that, in podocytes, the link between EZH2 and TxnIP is me- infancy, it is clear that these mechanisms exert effects that diated by H3K27me3 enrichment at the promoter region of are both cell type specific and responsive to the prevailing the transcription factor Pax6, which itself binds to the TxnIP milieu. promoter and regulates TxnIP expression. Pax6 is a transcrip- In summary, the histone methyltransferase EZH2 regu- tional regulator that is critically important for neurogenesis40 lates oxidative stress in diabetes. It does this by repressing and oculogenesis,41 and mutations within its encoding gene expression of the transcription factor Pax6 and consequently, lead to congenital iris hypoplasia, termed aniridia.42 To our the antioxidant inhibitor TxnIP (Figure 6). These findings knowledge, Pax6 has not previously been linked to podocyte expand the extent by which pathobiologic processes in di- (dys)function. However, these findings indicate that it may abetes complications may be influenced by epigenetic well have very important roles in these cells. Its gene is sepa- processes to include endogenous enzymatic antioxidant re- rated by only 750 kb from the gene encoding WT1 on the short pair mechanisms. arm of chromosome 11 in the human genome (approximately 450 kb apart on chromosome 2 in mice), and genetic deletions in this region can cause WAGR syndrome characterized by CONCISE METHODS Wilms tumor, aniridia, genitourinary anomalies, and intellec- tual disability (formerly known as mental retardation).43 Cell Culture Intriguingly, unlike in the setting of diabetes, we conversely Differentiated conditionally immortalized mouse podocytes were and unexpectedly observed a protective effect of DZNep when cultured as previously described.49 For pharmacologic EZH2 deple- it was administered to mice before IRI. Although this response tion, serum-starved cells were incubated with DZNep (5 mM; warrants further mechanistic investigation, it does serve to MedChem Express, Monmouth Junction, NJ) in DMSO or DMSO highlight the pervasive and most importantly, context-specific alone (0.1%; control) for 36 hours before supplementation of the effects that histone modifications and histone-modifying medium with 19.4 mM glucose (final concentration of 25 mM; high interventions can play in adult kidney (patho)physiology. In glucose) or maintenance under normal (5.6 mM) glucose conditions this study, we concentrated on the possible role of EZH2 in for an additional 12 hours. Mannitol (19.4 mM) served as the os- regulating oxidative stress in podocytes, whereas in other motic control. For shRNA–mediated gene knockdown or vector– settings, miR-101 may regulate endothelial function,44 EZH2 based gene overexpression experiments, podocytes were transfected has been associated with adverse cancer prognoses,45 and (Lipofectamine 2000; Life Technologies, Carlsbad, CA) with 2.5 mg TxnIP itself has been shown to function as a tumor suppres- sequence-specific shRNA targeting TxnIP (Origene, Rockville, MD) sor.46,47 These alternative actions should be borne in mind or EZH2 (Qiagen, Valencia, CA) or a DEST40 vector (Life Technol- when considering whether the pathway herein described is ogies) containing the mouse Trx gene with constitutive expression suitable for therapeutic targeting in patients. Furthermore, driven by a cytomegalovirus promoter (Origene). Cells were incu- despite consistency in the response to DZNep and EZH2 bated for 36 hours before supplementation of medium with normal shRNA in cultured cells and DZNep in diabetic rats, the ob- or high glucose for an additional 12 hours as already described. servation that H3K9me2 levels were also diminished by the Scrambled shRNA (Origene) was used as control. For inhibition of small molecule in cultured podocytes raises the possibility that miR-101, cells were incubated with a sequence–specific miR-101 in- the in vivo effects of DZNep may not be limited to the conse- hibitor (75 pM; Life Technologies) for 36 hours before exposure to quences of EZH2 depletion alone. normal or high-glucose conditions for 12 hours as already described. Although EZH2 has been most extensively studied in the For Pax6 knockdown, cells were transfected with sequence-specific context of development and neoplasia,48 its presence in podo- siRNA (100 nM) or scrambled siRNA (100 nM; Life Technologies) cytes and the physiologic sequelae of its depletion highlighted before incubation with DZNep (5 mM) or vehicle for 48 hours. For to us that the histone methyltransferase likely exerts different the determination of TxnIP expression in other renal cell types, functional effects in terminally differentiated adult Sprague–Dawley rat mesangial cells, NRK-52E cells, and MDCK cells. Accordingly, although we had focused our studies on cells were cultured as previously described.5 the regulation of one antioxidant pathway, we performed gene expression analyses with the purpose of (1) providing Immunoblotting an unbiased validation for the upregulation in TxnIP expres- Immunoblotting was performed on cultured cell extracts and whole- sion and (2) exploring the biologic processes most affected by kidney lysates with antibodies in the following concentrations: EZH2, EZH2 depletion. Interestingly, whereas a similar analysis 1:1000 (Cell Signaling Technology, Danvers, MA); TxnIP, 1:1000 highlighted a role for EZH2 in survival, differentiation, and (MBL International, Woburn, MA); H3K27me3, 1:1000 (Cell Sig- proliferation of cancer cells,9 EZH2depletioninpodocytes naling Technology); H3K4me3, 1:1000 (Novus Biologicals Canada (which are postmitotic) involved a qualitatively different set ULC, Oakville, ON, Canada); H3K9me2, 1:1000 (Abcam, Inc., of biologic processes primarily concentrated on the Cambridge, MA); H3K79me3, 1:1000 (Novus Biologicals Canada

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ULC); total histone H3, 1:1000 (Cell Signaling Technology); Pax6, 8-hydroxy-29-deoxyguanosine was measured by ELISA (Biotang, 1:1000 (Millipore, Billerica, MA); and b-actin, 1:5000 (Abcam, Inc.). Waltham, MA). Densitometry was performed using Image J software (version 1.47; National Institutes of Health). Study 3 Male C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) age 8 Intracellular ROS Measurement weeksoldwererandomized toreceiveeitherDZNep(1mg/kg)orPBSip. The fluorescent probe CFDA was used to measure intracellular ROS. Twenty-four hours later, mice were anesthetized (maximum dose of After incubation of cells with 10 mmol/L CFDA at 37°C for 15 minutes, 200 mg/kg ketamine and 10 mg/kg xylazine ip) and further randomized intracellular ROS levels were determined by measuring 10,000 events to bilateral clamping of the renal pedicles for 30 minutes or sham per sample by flow cytometer (FACS Calibur; BD Biosciences, San Jose, surgery50 (n=6–10 per group). After another 24 hours, blood was col- CA) after excitation with a 488-nm laser. Flow cytometry data were lected for measurement of plasma creatinine and BUN (Toronto Centre analyzed using CellQuest Pro software (BD Biosciences). for Phenogenomics, Toronto, ON, Canada), and kidney tissue was harvested for staining with hematoxylin and eosin. A tubular injury score Programmed Cell Death Detection was calculated by counting the percentage of tubules that displayed Podocyte programmed cell death was assessed using TUNEL. Cells with evidence of injury (cast formation, dilation, loss of the brush border, TUNEL-positive nuclei were counted at low magnification (3100) and and cell necrosis as follows: 0, no damage; 1, 0%–25% damaged expressed as a percentage of the total number of cells. Experiments were tubules; 2, 25%–50% damaged tubules; 3, 50%–75% damaged performed in triplicate. tubules; and 4, .75% damaged tubules)51 in 10 nonoverlapping fields (magnification, 3400)takenfromtheouterstripeoftheouter Quantitative Real–Time PCR RNA isolation from cell extracts was performed using TRIzol Reagent medulla. (Life Technologies). cDNA was reverse transcribed from 2 mgtotal RNA using reverse transcription enzyme (Roche Diagnostics, Laval, Study 4 QC, Canada) in the manufacturer’s buffer supplemented with 1 mmol/L In study 4, glomeruli were isolated by differential sieving52 from con- deoxynucleotide triphosphates and 2 mg random hexamers (GE trol male Sprague–Dawley rats and rats with 3 weeks of STZ-induced n n Healthcare Life Sciences, Mississauga, ON, Canada). Real-time PCR diabetes (control, =4; diabetes, =7) before immunoblotting for was performed using SYBR Green (Applied Biosystems, Foster City, EZH2, Pax6, and TxnIP. CA) on an ABI Prism 7900HT Fast PCR System (Applied Biosystems). All experimental procedures adhered to the guidelines of the ’ Primers for EZH2 were from Qiagen. Other primer sequences were Canadian Council on Animal Care and were approved by St. Michael s from ACGT Corp. (Toronto, ON, Canada) and are shown in Supple- Hospital Animal Care Committee. mental Table 6. Primer sequences used for the determination of TxnIP expression in mesangial cells, NRK-52E cells, and MDCK cells were as Electron Microscopy previously described.5 Experiments were performed in triplicate, and Transmission electron microscopy was performed on kidney cortical tissue data analyses were performed using the Applied Biosystems Compar- from four rats from each group (Philips CM100 Transmission Electron Microscope; Newcastle University). Representative micrographs from ative CT Method. each animal were examined using a semiquantitative technique to assess In Vivo Studies podocyte morphology and identify podocyte abnormalities (adsorption Study 1 droplets, vacuoles, or pseudocysts) per area glomerulus as previously Male Sprague–Dawley rats (Charles River Laboratories, Wilmington, described.53 MA) age 8 weeks old were randomized to receive either DZNep (1 mg/kg) in PBS or PBS alone (vehicle) by ip injectionondays1and4andfollowed Immunohistochemistry for a total of 5 days (n=4 per group). Immunohistochemistry was performed on 4-mm rat kidney sections. Primary antibodies were used in the following concentrations: TxnIP, Study 2 1:800 (MBL International) and JG12, 1:1000 (Bender MedSystems, Male Sprague–Dawley rats were randomized to receive either STZ (60 Vienna, Austria). Glomerular immunostaining was quantified using mg/kg; Sigma-Aldrich, St. Louis, MO) in 0.1 Mcitrate buffer (pH 4.5) or Aperio ImageScope software (Aperio Technologies Inc., Vista, CA) in citrate buffer alone by tail vein injection after an overnight fast. Animals approximately 30 glomeruli per kidney section. Data are expressed as were then further randomized to receive either DZNep (1 mg/kg ip fold change compared with normoglycemic, vehicle–treated rats. Tu- twice weekly) or vehicle for 3 weeks (n=12 per group). Diabetic rats bule epithelial cell death was determined as the number of positively received a thrice weekly subcutaneous injection of insulin (1–4 units staining nuclei in 10 randomly selected cortical fields (magnification, subcutaneously; Humulin N isophane; Eli Lilly Canada, Toronto, ON, 3400) from eight rats per group after TUNEL.22 Canada). Systolic BP was measured by tail cuff plethysmography using a 5 noninvasive BP system as previously described. Hemoglobin A1c was Mesangial Matrix Index measured using A1cNow+ (Bayer, Sunnyvale, CA). Urine A minimum of 50 glomeruli were examined in periodic acid– protein excretion was measured by the benzethonium chloride method Schiff–stained kidney sections from each rat. Mesangial matrix after housing rats individually in metabolic cages for 24 hours. Urinary was assessed as previously described.54

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Promoter Reporter Assay aligned sequence read plots generated using the Integrated Podocytesweretransfectedwithaluciferasereporterunderthecontrolof Genome Browser.58 the TxnIP promoter,pGL3B-1081(giftfromClark Distelhorst;Addgene 55 m plasmid 18759). Cells were incubated with DZNep (5 M) or vehicle Microarray for 48 hours before determination of luciferase activity with a reporter In total, 300 ng mRNA was hybridized to the Illumina Mouse WG-6 assay system (Promega, Madison, MI). v2.0ExpressionBeadChip.IlluminaGenomeStudiosoftwarewasused for average normalization (with background subtraction) and dif- ChIP and ChIP Sequencing ferential gene analysis (data available at Gene Expression Omnibus ChIP analysis was performed using the Magna ChIP Kit (EMD accession no. GSE60038). In average normalization, sample intensi- Millipore). Briefly, mouse podocytes were cultured as described. After ties were scaled (to the average intensity of the sample) and crosslinking and sonication, the sheared chromatin was immuno- background subtracted before scaling. The false discovery rate was precipitated overnight using antibodies against H3K27me3 (1:50; Cell controlled using the Benjamini–Hochberg method. The Illumina Signaling Technology), Pax6 (1:100; EMD Millipore), or normal custom model with the default false discovery rate correction was rabbit IgG (1:50; control; Santa Cruz Biotechnology, Dallas, TX). used for differential gene expression. Genes for downstream anal- Then, it waswashed, reverse crosslinked,and treated withproteinase K yses were filtered to include only those with differential expression to obtain purified DNA fragments. Quantitative real–time PCR was analysis–adjusted P values of ,0.05. Partek Genomics Suite (Partek performed using a set of custom primers obtained from IDT Inc. Inc., St. Louis, MO) was used for hierarchical clustering of samples; (Coralville, IA) specific for a sequence of the mouse Pax6 or TxnIP Pearson dissimilarity and average linkage were used to cluster both promoter. The sequences of the PCR primers are found in Supple- genes and samples. Lists of differentially expressed transcripts were mental Table 6. uploaded to the web–based DAVID Bioinformatics Resources 6.7 For ChIP sequencing, podocytes were treated with vehicle or (NIAID/National Institutes of Health) Functional Annotation DZNep (5 mM) for 48 hours before ChIP as described. After treat- Tool. 59,60 Results were visualized using Java Treeview (http://jtreeview. ment, cells were harvested and processed following the Magna sourceforge.net). ChIP Kit protocol (Millipore). Sheared DNA from whole-cell lysates was immunoprecipitated using an H3K27me3 antibody Statistical Analyses (1:50; Cell Signaling Technology). Whole–cell lysate sheared Data are expressed as means6SEMs. Statistical significance was de- DNA from the same treatment groups was considered as input. termined by one-way ANOVAwith Fisher’s least significant difference For ChIP–sequencing library construction, 10 ng ChIP DNA or test or t test where appropriate. Statistical analyses were performed input DNA was used after purification with AMPure XP Beads using GraphPad Prism software (version 6.02; GraphPad Software (Beckman Coulter Inc., Mississauga, ON, Canada). Libraries Inc., San Diego, CA). from the immunoprecipitated and whole–cell lysate DNA were prepared using the NEBNext ChIP-Seq Library Prep Reagent Set for Illumina (New England BioLabs Inc., Ipswich, MA). In brief, ACKNOWLEDGMENTS immunoprecipitated DNA was first end repaired using the End-It Repair Kit (Epicentre, Madison, WI) followed by dA tailing of end- The authors thank Youan Liu and M. Golam Kabir for excellent repaired DNA using the NEBNext dA-Tailing Reaction Mixture technical assistance and Kryski Biomedia for artwork. (New England BioLabs Inc.) and ligating to custom adapters These studies were supported by biomedical research grants from with NEBNext Multiplex Oligos for Illumina (New England BioLabs the Kidney Foundation of Canada and Canadian Diabetes Association Inc.). Fragments of 150–700 (+120 bp) bp of adaptor-ligated DNA Operating Grant OG-3-14-4502 (to A.A.). F.S.S. was supported by a were size selected using AMPure XP Beads followed by ligation– Banting and Best Diabetes CentreFellowship in DiabetesCare(funded mediated PCR amplification of the adaptor-ligated DNA using by Eli Lilly Canada). S.M. is supported by a Canadian Diabetes As- Phusion DNA Polymerase (New England Biolabs Inc.). On library sociation Post–Doctoral Fellowship. M.A. was supported by summer preparation, the samples were sequenced on a HiSEquation 2500 research fellowships from the Ontario Genomics Institute and the System (Illumina, San Diego, CA) with a 23 100-bp end (The Endocrine Society. G.G. was supported by a Charles Hollenberg Centre for Applied Genomics, The Hospital for Sick Children, Summer Studentship from the Banting and Best Diabetes Centre. Toronto, ON, Canada; data available at Gene Expression Omnibus K.A.C. was supported by a New Investigator Award from the accession no. GSE69610). Sequence data quality was assessed using Canadian Institutes of Health Research. A.A. was support by a FastQC v.0.11.2, and adaptors were trimmed using Trimmomatic Reuben and Helene Dennis Scholar Award from the Banting and Best v0.32. Sequenced reads were aligned to the mouse reference ge- Diabetes Centre and a Clinician Scientist Award from the Canadian nome (UCSC mm10) for each ChIP-sequencing experiment in Diabetes Association. mouse podocytes using Bowtie2 v2.2.56 Peaks were identified using Portions of this work were presented in abstract form at the Annual both MACS software (version 1.4.2) with parameter values (band Scientific Meeting of the American Diabetes Association (Chicago, IL, with: 100; m: 10,20; p: 1e-5; on-auto: TRUE; w: TRUE; shift size: June 21–25, 2013) and the Annual Scientific Meeting of the European 300) and visualization using the UCSC Genome Browser (https:// Diabetic Nephropathy Study Group (London, United Kingdom, May genome.ucsc.edu/) and CisGenome57 (default parameters) with 16–17, 2014).

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2034 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 2021–2034, 2016