Full Reviews Evidence for the involvement of

Nephrol Dial Transplant (2014) 29: i1–i8 doi: 10.1093/ndt/gft361 Advance Access publication 17 September 2013

Full Reviews Evidence for the involvement of epigenetics in the progression of renal fibrogenesis Björn Tampe and Michael Zeisberg Department of Nephrology and Rheumatology, Göttingen University Medical Center, Georg August University, Göttingen, Germany

Correspondence and offprint requests to: E-mail: [email protected]

A B S T R AC T

H I S T O N E M O D I F I C AT I O N S

Epigenetics are omnipresent in eukaryotic cells and influence cell differentiation and maintenance of cell metabolism in health and disease. Here, we discuss how the ‘second genetic code’ impacts the fate of the injured kidney. We provide a glimpse of how recent insights into epigenetic mechanisms of chronic kidney disease might lead to novel diagnostic and therapeutic tools.

In mammals, DNA is spun around histone protein cores, which serve to physically strengthen it, preventing DNA strand breaks as well as impacting replication and gene expression [1]. Each of these so-called ‘histone cores’ consists of 147 base pairs of genomic DNA wrapped around a protein octamer containing dimers of histones H2A, H2B, H3 and H4 (Figure 1) [1]. Each of these histones is a product of distinct, highly conserved genes, existing as multiple copies in the human genome. There are two principal organizational states of chromatin, which are referred to as ‘euchromatin’ (which is structurally loose to allow RNA polymerases and transcriptional factors to bind) and ‘heterochromatin’ (which is tightly packed and associated with transcriptional inactivation) [1]. The organization of chromatin is variable and depends on several factors, prominently cell cycle stage [1]. As the cell enters mitosis, chromatin has to be tightly packaged to prepare for cell division. During interphase, transcriptional activation is associated with the establishment of ‘euchromatin’, forming nucleosomes that are interconnected by sections of linker DNA (20–60 base pairs) and the linker histone H1 (Figure 1) [1]. Histone proteins determine local chromatin formation and transcriptional activation by their post-translational modifications like methylation, phosphorylation, acetylation and SUMOylation affecting histone function and transcriptional activity [5]. Histone post-translational modifications are categorized as intrinsic, extrinsic and effector-transduced mechanisms [6–8]. Intrinsic mechanisms refer to post-translational cleavage or attachment of covalent chemical groups to histone proteins [6]. These modifications directly influence the physiological activities of nucleosomes, such as DNA binding, mobility, conformation or stability [6]. Extrinsic effects alter

Keywords: epigenetic, histone modification, hydroxymethylation, kidney fibrosis, methylation

INTRODUCTION In its widest meaning, the term ‘epigenetics’ refers to chromatin modifications that regulate gene expression. The main components of chromatin are histone proteins and the DNA wrapped around them, and epigenetic modifications can involve both of them, referred to as histone modifications and DNA methylation [1, 2]. Both types of modifications can stably influence the transcription of multiple genes and can be maintained from one cell generation to the next without changing the primary nucleotide sequence (in contrast to genomic polymorphisms or mutations) [3]. Due to the fact that epigenetics stably influence gene expression even after the initial mechanism has passed, they have been referred to as the ‘second genetic code’ [4]. Due to the fact that epigenetics are omnipresent in eukaryotic cells and influence cell differentiation and maintenance of cell metabolism, its role in health and disease is increasingly recognized. Here, we review epigenetics and their implications on the progression of chronic kidney disease. © The Author 2013. Published by Oxford University Press Downloaded from https://academic.oup.com/ndt/article-abstract/29/suppl_1/i1/1871763 on behalf of ERA-EDTA. All rights reserved. by guest on 05 February 2018

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F I G U R E 1 : Histone code in mammalian cells. The octameric

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histone core consists of 147 base pairs of genomic DNA wrapped around dimers of histones H2A, H2B, H3 and H4. These histone cores are interconnected by sections of linker DNA (20–60 base pairs) and the linker histone H1 forming nucleosomes.

inter-nucleosomal conformation, chromatin remodelling and structure [6]. Effector-transduced mechanisms refer to altered enzymatic activities of non-histone components and intra- or inter-nucleosomal dynamics [6]. These modifications appear in a regulated, non-random manner, leading to the hypothesis that post-translational histone alterations constitute a distinct ‘histone code’ regulating gene expression [1, 8]. Most histone modifications have been described on histone tails becoming adhesive molecules to recruit and bind specific complexes to modulate gene transcription [5, 9]. Depending on the modified residue, these histone modifications can transduce transcriptional repression or induction. For example, trimethylation of H3 at lysine 27, namely H3K27me3, is a strong repressor of transcription by attracting chromodomain-containing proteins and HP1 [5, 9]. In contrast, trimethylation of H3 at lysine 3, namely H3K3me3, binds certain proteins containing a bromodomain motif associated with transcriptional induction [5, 9]. In addition to the histone tail, the histone core undergoes post-translational modifications [10]. Since the histone core is tightly wrapped with DNA, post-translational modifications by remodelling complexes must weaken the interaction between DNA and its nucleosome in order to expose the amino acid residue [11, 12]. Among histone modifications, histone acetylation has been best studied in the context of chronic kidney disease (or any pathologies for that matter), because histone acetylation can be interfered with by using pharmacological inhibitors of histone deacetylases (HDACs) [11, 12]. In general, histone acetylation is a major mechanism in the regulation of the chromatin state [11, 12]. For transcriptionally active genes, acetylation restores the positive charge of lysine residues loosening histone interactions with negatively charged DNA [11, 12]. This euchromatin formation gives access for transcriptional factors contributing to transcriptional induction. In contrast, deacetylation of histone lysine residues causes transcriptional silencing [11, 12]. Histone acetylation is balanced by acetyltranferases (HATs) and HDACs [11, 12]. Importantly, HDACs can be targeted through various HDAC inhibitors, which generally prevent HDAC-induced transcriptional silencing [11, 12]. In addition, non-histone proteins can be

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modified by HATs and HDACs, e.g. transcriptional factors, affecting their binding properties [13]. Depending on their sequence similarity and cofactor interactions, HDACs can be classified into four classes with different members: Class I (HDAC1, 2, 3 and 8) are nuclear enzymes widely expressed in different tissue types [14]. In contrast, Class II (HDAC4, 5, 6, 7, 9 and 10) and IV (HDAC11) are predominantly located within the cytoplasm expressed in a tissue-specific manner [14]. Class III of HDACs belongs to the sirtuin family with seven as of yet known isoforms (SIRT1–7) that differ in their subcellular localizations, substrate specificities and functions [15]. In the adult kidney, all members of Class I and II HDACs are expressed [14]. Specific functions of HDAC members in the progression of kidney disease, however, are only incompletely understood, and current understanding is mostly based on therapeutic effects of more- or less-specific HDAC inhibitors [16–19]. In injured kidneys, HDAC1, 2, 5 and 6 are consistently induced contributing to decreased histone acetylation [16]. HDAC2 and 5 have been suggested to contribute transcriptional suppression of reno-protective BMP7 [17–19]. HDAC inhibition through the administration of HDAC inhibitors, such as trichostatin A (TSA) or dexmedetomidine, have been shown to be anti-fibrotic in experimental fibrosis models through rescuing BMP7 expression [17–19]. While most studies which demonstrated a role of histone deacetylation in the progression of renal fibrosis utilized TSA, an unselective HDAC Class I/III inhibitor, administration of MS-275, a selective Class I HDAC inhibitor, ameliorated fibrosis through inhibition of transforming growth factor (TGF)beta1 signalling and rescued G2/M cell cycle arrest of tubular epithelial cells [16, 20–23]. Administration of a specific HDAC6 inhibitor, tubacin, ameliorated experimental fibrosis through the normalization of epidermal growth factor receptor (EGFR) endocytic trafficking and degradation in renal epithelial cells, highlighting a specific contribution of HDAC6 to the progression of chronic kidney disease [24]. Similarly, administration of vorinostat, the first HDAC inhibitor approved for clinical application, ameliorated experimental renal fibrosis through the normalization of EGFR-mediated signalling [25]. Histone modifications that contribute to the progression of renal fibrosis are not limited to deacetylation. Phosphorylation of histone H3 contributes to the incapability of tubular epithelial cells to regenerate and proliferate in chronic kidney disease due to G2/M phase arrest [26]. Histone methylation is a common histone modification that is well studied in the context of histone marks indicative of transcriptional activity (i.e. tri-methylated lysine 4 involving histone H3/H3K4me3) or transcriptional suppression (i.e. tri-methylated lysine 27 involving histone H3/H3K27me3) [27]. Expression of pro-fibrotic TGF-beta1 correlates with histone methylation marks [28]. Conditional ablation of pax transactivation domain-interacting protein, a constituent of the histone H3 lysine 4 (H3K4) methyltransferase complex in glomerular podocytes, caused podocyte effacement and chronic kidney failure in mice [29]. This highlights the crucial role of histone methylation in kidney health and disease. Not only do post-translational modifications occur within the histone core, histone components forming the histone core

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methylcytosines (which is referred as ‘DNA methylation’) mediated by DNA methyltransferases [2]. In this enzymatic reaction, a methyl group is added to the carbon 5 of the pyrimidine ring generating 5-methylcytosine (5-mC) [2]. The methylation status of these CpG islands regulates gene expression by affecting the binding of transcriptional factors and facilitation of heterochromatin formation [43]. Depending on its physiological status and function, each cell provides its certain cluster of CpG methylation depending on the genes transcriptionally silenced or activated. Alterations can be described for increased CpG methylation as ‘hypermethylation’ and ‘hypomethylation’ for decreased CpG methylation [2, 44]. For most genes, hypermethylation transduces transcriptional silencing, whereas hypomethylation can restore gene expression of normally silenced genes (Figure 2) [2, 44]. DNA methylation physically alters transcriptional factor-binding affinity, and the predominant mechanism is mediated by methylated DNA-binding proteins blocking transcriptional factor binding [45]. These proteins can be classified into MBDs (methyl-CpG-binding domain proteins), UHRF proteins (ubiquitin-like, containing plant homeo domain (PHD) and really interesting new gene (RING) finger domains) and zinc finger proteins [45]. The MBD family

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itself can also contribute to the intrinsic and extrinsic properties by forming specialized chromatin structures affecting transcriptional activity [30]. The highly evolutionary conserved variant, H2A.Z, is present in all eukaryotes with a large variety of different functions. Its sequence shows an identity to the major H2A of only ∼60% suggesting distinct functions of H2A.Z [30, 31]. H2A.Z is only present in the transcriptionally active macronucleus, but not in the inactive micronucleus highlighting its role in transcriptional regulation [32]. Interestingly, H2A.Z has been found to mediate both transcriptional activation and repression by affecting nucleosome mobility and positioning [33, 34]. As a consequence, incorporation of H2A.Z differentially regulates binding affinities of activating and repressing nuclear factors to their target motif in association with regulatory regions like enhancers, insulators and heterochromatin formation [35]. In the context of kidney injury, H2A.Z incorporation is increased, corresponding to the activation mark H3K4me3 at genes encoding for TGF-beta1 and Type I collagen [36]. While substitution of H2A.Z with generic H2.A correlates with kidney disease and activation of pro-fibrotic genes, little is yet known about the stimuli that recruit H2A.Z to affected genes [36]. Histone H2A.X is another histone H2A variant, which substitutes for up to 50% of all H2.A proteins [37]. H2A.X is best known as a marker for DNA damage [37]. After DNA doublestrand breaks, H2A.X becomes serine phosphorylated at position 139 (S139), which is referred to as γH2A.X [37]. A key function of γH2A.X S139 phosphorylation is to generate a docking site for DNA damage repair factors near or at DNA double-strand breaks [37]. These factors include the mediator of DNA damage checkpoint protein 1 (MDC1), which has been shown to bind directly to phosphorylated S139 of γH2A. X [38, 39]. The interaction between MDC1 and γH2A.X is only possible if a second residue of γH2A.X, tyrosine at position 142 (Y142), is dephosphorylated by specific tyrosine phosphatases of EYA proteins [38, 39]. When Y142 remains phosphorylated, binding of DNA damage repair factors is reduced and the pro-apoptotic c-Jun N-terminal kinase (JNK) is recruited instead [40]. The chronically injured kidney is associated with the substantial accumulation of γH2A.X-positive cells, possibly indicating double-strand breaks through oxidative stress [41]. In summary, chronic kidney injury is associated with robust histone modifications, which can either involve histone side chains or replacement of generic histone isoforms. Because of the availability of HDAC inhibitors, histone acetylation is currently the best-established histone modification in the context of kidney fibrosis, but other modifications are just as likely to be involved.

C P G M E T H Y L AT I O N

F I G U R E 2 : Effect of CpG methylation and hydroxymethylation on

In genomic DNA, cytosine and guanine are linked with a single phosphate to form a complex called CpG dinucleotide [2]. Enrichment of these so-called CpG islands (CpGs) can especially be observed in predicted promoter regions of ∼50– 60% of all human genes (CpG island promoters) [42]. Within these CpG islands, cytosines can be converted into

transcriptional activity. (A) Gene expression is active when cytosines within promoter CpGs are unmethylated (yellow). (B) In mammals, three members of DNMTs (DNMT1, DNMT3A and DNMT3b) are known to actively convert cytosine into 5-mC (red) associated with transcriptional repression. (C) The TET family members TET1, TET2 and TET3 can convert methylated (red) to hydroxymethylated (blue) cytosines inducing transcriptional activity.

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Epigenetics of renal disease

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consists of 11 members characterized by a methyl-CpGbinding domain, which is highly conserved, including MeCP1 and MeCP2, MBD1–6, together with Kaiso [45, 46]. MeCP1 is a protein complex including MBD2 and the chromatin remodelling complex NuRD/Mi2 targeting nucleosomal DNA [47]. MeCP2 is characterized by two important domains, the methyl-binding and transcriptional-repression domain silencing transcription through the recruitment of a Sin3A repressor complex after binding to a single methylated CpG [48, 49]. UHRF1 binds to methylated CpG dinucleotides and recruits transcriptional repressors, e.g. DNA methyltransferase 1 (DNMT1) or HDAC1 [50–52]. DNMT1 maintains DNA methylation of methylated CpG dinucleotides negatively influencing promoter activity and gene expression by transcriptional repression [53]. In this context, HDAC1 promotes heterochromatin formation of these genomic regions [53]. Not only promoters, but also gene bodies, can be methylated affecting intronic and exonic regions [54]. In contrast to CpG methylation associated with transcriptional repression, gene body methylation occurs in actively transcribed genomic regions [55]. RNA polymerases attract DNMTs and methylation can be observed as a consequence of transcriptional activation [56, 57]. Furthermore, intronic methylation can regulate alternative splicing by altered affinity of DNA-binding proteins depending on the methylation status [58]. In summary, DNA methylation is the prototypical epigenetic mechanism as it is the most stable and passed on through numerous cell generations [3]. Depending on the specific site of CpG methylation within the genome, DNA methylation influences transcriptional regulation and activation. In kidney fibrosis, fibroblast activation and transdifferentiation into myofibroblasts have been linked to hypermethylation and transcriptional silencing of a subset of genes [59]. In a genome-wide screen, 12 genes (DLG2, Enc, EYA1, Lzts1, HIPK1, HIPK2, HIPK3, Lrnf2, Odd1, Pax3, RASAL1 and Zu5) could be identified as hypermethylated in all primary myofibroblasts out of fibrotic kidneys compared with normal fibroblasts out of healthy kidneys [59]. For example, RASAL1 encodes for the rasGAP-like protein 1 (RASAL1) that inactivates Ras-GTP signalling [59]. In experimental kidney injury, acute renal damage and chronic progressive fibrosis are associated with transcriptional repression of Rasal1 [59]. While spontaneous kidney regeneration after acute injury is associated with normalized Rasal1 expression, Rasal1 expression remains silenced in chronic progressive fibrosis due to Rasal1 hypermethylation [59]. Decreased Rasal1 expression levels cause increased RasGTP signalling contributing to fibroblast activation and kidney fibrosis [59]. The pro-fibrotic cytokine TGF-beta1 is up-regulated in tissue fibrosis of several organs and causes fibroblast activation [59]. In cell culture, short-term exposure to TGFbeta1 causes transient transcriptional repression of Rasal1 without Rasal1 promoter hypermethylation [59]. Long-term exposure to TGF-beta1 induces Rasal1 repression by Rasal1 promoter hypermethylation, which is also persistent if TGFbeta1 is removed [59]. Transcriptional repression of Rasal1 by epigenetic mechanisms as part of fibroblast activation leading to kidney fibrosis is also present in the liver, suggesting a common role in tissue fibrosis beyond the kidney [60].


D N A M E T H Y LT R A N S F E R A S E S The conversion of cytosines into methylcytosines is facilitated by a class of enzymes referred to as DNMTs [53]. In humans, four active DNMTs have been identified: DNMT1, 3A, 3B and 3L [53]. DNMTs are commonly separated into de novo methyltransferases and those maintaining DNA methylation and there is evidence for both, supporting and refuting this classification. Support for this classification scheme originally came from the observation that embryonic stem (ES) cells retained de novo methylation after knockdown of Dnmt1 implicating its role in maintaining DNA methylation [53]. Based on these results, continued search for new DNMT members discovered the not yet known DNMT3 family [61]. Further support for the de novo/maintenance concept came from observations that the homozygous deletion of Dnmt3a and Dnmt3b in murine ES cells did not affect pre-existing DNA methylation, whereas the deletion of Dnmt1 led to a one-third reduction [62]. In contrast to this concept, increased de novo methylation could be observed after over-expression of DNMT1 in human fibroblasts [63]. Different in vitro studies regarding the affinity to unmethylated (de novo methylation activity) and hemimethylated (maintenance of methylation) double-stranded DNA could show that DNMT1 is capable of both with a preference for hemimethylated DNA that ranges between 15- and 134-fold [64, 65]. Probable reasons for this discrepancy may be the quality of the enzyme depending on the isolation procedure and that its in vivo activity differs from that observed in vitro. It is possible that, in the presence or absence of interacting nuclear factors, such as p21, proliferating cell nuclear antigen (PCNA), ubiquitin-like, containing PHD and RING finger domains 1 (UHRF1) and MBD proteins, all members of DNMTs possess both de novo and maintenance functions [66, 67]. Due to the fact that DNMT1 is the predominant methyltransferase in mammalian cells, it is currently viewed as pivotal in both de novo and maintenance of DNA methylation [68, 69]. Respective to the involvement of DNMTs in kidney fibrosis, it has been shown that Dnmt1 is induced in experimental renal fibrosis, whereas Dnmt3a and Dnmt3b remained unregulated [59]. Further insights into the contribution of Dnmt1 to the progression of renal fibrogenesis were provided by analysis of Dnmt1 heterozygous knockout mice, which display decreased expression levels of Dnmt1 [59]. As hypermethylation can be observed in kidney fibrosis associated with an increase in Dnmt1, these mice show ameliorated aberrant promoter methylation [59]. This is associated with a reduction of renal tubulointerstitial fibrogenesis in experimental fibrosis [59]. Other than DNMT1, no other member of the DNMT family has yet been identified to be altered in kidney fibrosis, highlighting its predominant role in aberrant hypermethylation in context of chronic progressive kidney disease.

D N A H Y D R O X Y M E T H Y L AT I O N While DNA methylation marks are prominently known for their stability, as they can be passed on over multiple cell

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EPIGENETIC THERAPIES IN RENAL DISEASE As histone modifications and DNA hypermethylation are involved in the progression of kidney fibrosis, pharmacological modification and inhibition of aberrant epigenetic modifications have been shown to attenuate fibrogenesis and to be beneficial for the kidney. Several clinical trials are currently testing pharmacological therapies against aberrant epigenetic modifications in cancer by pharmalogical inhibition of HDACs (Table 1) and erasure of aberrant methylation (Table 2). Future studies have to elucidate their efficiency in the treatment of renal disease. Treatment of aberrant histone acetylation in experimental kidney fibrosis with TSA, a specific inhibitor of Class I and II HDACs, attenuates intra-renal inflammation and tubulointerstitial fibrosis in mice challenged with different models of experimental kidney fibrosis (Table 3)


[16, 84]. Furthermore, treatment with TSA inhibits apoptosis and epithelial-to-mesenchymal transition programming in human tubular epithelial cells in vitro [85]. Other Class I and II HDAC inhibitors like phenylbutyrate and valproic acid could have been shown to be beneficial in the streptozotocininduced diabetic nephropathy, doxorubicine-induced nephropathy and experimental kidney fibrosis due to administration of TGF-beta1 (Table 3) [21, 23, 86]. In the erasure of aberrant methylation, pharmacological demethylation agents are tested in several clinical trials, most of them in cancer therapies (Table 2). 5-Azacytidine and its derivate 5-Aza-2-deoxycytidine are incorporated as 5-Aza-dCTP

Table 1. Clinical trials of pharmacological inhibitors of HDACs listed by the US National Institutes of Health Name

Target

# of clinical trials

Disease

Belinostat

HDAC Class I HDAC Class II HDAC Class IV

32

Entinostat

HDAC Class I

23

Givinostat

HDAC Class I HDAC Class II

5

MGCD0103

HDAC Class I

12

Panobinostat

HDAC Class I HDAC Class II HDAC Class IV

114

Phenylbutyrate


40

Romidepsin

HDAC Class I

53

Tacedinaline

HDAC Class I

3

Valproic acid

HDAC Class I

257

Vorinostat


214

Carcinomas Leukaemias Lymphomas Sarcomas Carcinomas Leukaemias Lymphomas Sarcomas Crohn’s disease Leukaemias Lymphomas Muscular dystrophies Rheumatic diseases Carcinomas Leukaemias Lymphomas HIV infection Carcinomas Graft-versus-host disease Leukaemias Lymphomas Sarcomas HIV infection Carcinomas Leukaemias Lymphomas Carcinomas Leukaemias Lymphomas Sarcomas Carcinomas Lymphomas HIV infection Carcinomas Leukaemias Lymphomas Sarcomas HIV infection Carcinomas Graft-versus-host disease Leukaemias Lymphomas Sarcomas


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generations, they are not permanent but can be erased (demethylated) [70]. In general, DNA demethylation can occur through passive or active mechanisms. Passive loss of DNA methylation can only be observed in dividing cells during replication, when marks on hemimethylated DNA are not copied to the newly synthesized strand [71]. Active DNA demethylation involves the recruitment of DNA repair mechanisms with subsequent nucleotide excision [70]. The mechanism that directly recognizes and cleaves the methyl group of 5-mC in aberrant DNA methylation is not yet known [70]. The most prominent active demethylation mechanism involves enzymatic oxidation of 5-mC to 5-hydroxymethylcytosine (5-hmC) to activate DNA repair and nucleotide replacement as part of epigenetic gene regulation [72]. In mammals, three known members of the 10–11 translocation (TET) protein family as yet identified are able to convert 5-mC into 5-hmC: TET1, -2 and -3 [73, 74]. Somatic mutations in the TET1 and TET2 genes are associated with aberrant CpG island promoter methylation in haematopoietic malignancies [75]. While CpG island promoter methylation causes transcriptional silencing, hydroxymethylation is associated with increased gene expression—even more than compared with the unmethylated status (Figure 2) [76]. It has been shown that all members of TET proteins have distinguished functions in embryogenesis [77–79]. Knockout experiments highlight their pivotal role in ES cell maintenance and differentiation, which requires demethylation of Oct4, Nanog and Sox2 [77–79]. Although all members of the TET family contain a highly conserved C-terminal catalytic domain, only TET1 and TET3 are characterized by a CXXC domain for DNA binding [80]. Such CXXC domains can also be found in DNA-binding proteins specifically targeting CpGs (e.g. DNMT1) [80–82]. Beside their role in stemness and pluripotency, TET family members are also highly expressed in adult tissue including the kidney [83]. In adult tissue, the distinct functions of each member of TET proteins remain unknown. In the context of kidney fibrosis, the role of TETmediated endogenous DNA demethylation mechanisms in the homeostasis of epigenetic modifications has not been described yet.

Table 2. Clinical trials of pharmacological erasure of aberrant methylation listed by the US National Institutes of Health Name

Target

# Clinical trials

Disease

5-Azacytidine

DNMTs DNA/RNA incorporation

243

Decitabine

DNMTs DNA/RNA incorporation

132

Hydralazine

DNMTs

30

Carcinomas Graft-versus-host disease Leukaemias Lymphomas Sarcomas Carcinomas Leukaemias Lymphomas Sarcomas Carcinomas Hypertension Leukaemias

Table 3. Studies of pharmacological modulation of the epigenome in experimental kidney fibrosis Name

Experimental model

Effect

5-Azacytidine

Nephrotoxic serum nephritis [59] Uninephrectomy-induced glomerulopathy [88] Sepsis-induced kidney injury [17] Radiocontrast-induced nephropathy [89] Ischaemia–reperfusion injury [90] Unilateral ureteral obstruction [20] Streptozotocin-induced diabetic nephropathy [86] Doxorubicine-induced nephropathy [21] Unilateral ureteral obstruction [16, 22] TGF-beta1-induced nephropathy [23] Streptozotocin-induced diabetic nephropathy [23] Nephrotoxic serum nephritis [91] Polycystic kidney disease [24] Doxorubicine-induced nephropathy [21] TGF-beta1-induced nephropathy [23] Streptozotocin-induced diabetic nephropathy [23]

DNA demethylation Klotho demethylation BMP7 induction Decreased tubular necrosis JAK/STAT inhibition

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5-Aza-2deoxycytidine Dexmedetomidine

MS-275 Phenylbutyrate Trichostatin A

Tubacin Valproic acid

TGF-beta1/EGFR inhibition ER stress/p-JNK inhibition Less macrophage infiltration Caspase-3 inhibition TGF-beta1/ROS inhibition TGF-beta1/ROS inhibition BMP7 induction EGFR inhibition Less macrophage infiltration TGF-beta1/ROS inhibition TGF-beta1/ROS inhibition

Since modifications of the epigenome are a more or less general process, pharmacological agents show several side effects. Regarding normalization of aberrant promoter methylation, 5-azacytidine and its derivate 5-Aza-2-deoxycytidine are randomly incorporated affecting methylation of not only aberrant methylated genes. Due to DNA intercalation and incorporation, both drugs are cytotoxic and long-term effects of genome-wide demethylation are not yet available. In recent years, an increasing number of selective HDAC inhibitors have been developed [11, 12]. These should show fewer side effects due to the more specific disruption of HDAC substrates, but long-term data are not available yet. Since TET-mediated hydroxymethylation has been identified as an endogenous mechanism of active DNA demethylation to restore gene expression in stemness and pluripotency, nothing is known about its role in kidney repair and fibrogenesis but this deserves future attention.

PERSPECTIVES Both CpG island promoter methylation and histone modifications impact the progression of experimental chronic kidney disease. Due to the insufficiency of genetics to explain the heterogeneity of progression rates among individual patients, epigenetics may present a superimposed mechanism. While validation in large clinical trials is still lacking, use of epigenetic marks as a diagnostic tool for individual risk stratification is an attractive concept. Furthermore, modulation of the epigenome might provide a therapeutic approach, which targets chronic fibrogenesis at its root. Recent advances of epigenetic therapies in the cancer field provide hope that such epigenetic therapies (such as the correction of CpG methylation through the administration of 5-azacytidine) might become available for clinical use in patients with chronic kidney disease in the future.

AC K N O W L E D G E M E N T S M.Z. is supported by DFG grant ZE523/2-1, ZE523/3-1 and the Genzyme GRIP Award. B.T. is supported by the ‘seed funding research program’ of the Faculty of Medicine, GeorgAugust-University Göttingen.

C O N F L I C T O F I N T E R E S T S TAT E M E N T The authors declare no conflict of interest.

cytidine analogues into genomic DNA and form a covalent complex with DNMT1 [87]. This results in an irreversible inhibition of DNMT1 and genome-wide demethylation after cytosine excision. In experimental renal fibrosis, treatment with the demethylation agent 5-azacytidine rescues kidney morphology and attenuates tubulointerstitial fibrosis in folic acid-induced nephropathy and uninephrectomized mice (Table 3) [59, 88].


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Received for publication: 18.6.2013; Accepted in revised form: 15.7.2013