Cell Tissue Res DOI 10.1007/s00441-017-2588-x

REVIEW

Epigenetics of kidney disease Nicola Wanner 1,2,3

&

Wibke Bechtel-Walz 1,3

Received: 31 December 2016 / Accepted: 15 February 2017 # Springer-Verlag Berlin Heidelberg 2017

Abstract DNA methylation and histone modifications determine renal programming and the development and progression of renal disease. The identification of the way in which the renal cell epigenome is altered by environmental modifiers driving the onset and progression of renal diseases has extended our understanding of the pathophysiology of kidney disease progression. In this review, we focus on current knowledge concerning the implications of epigenetic modifications during renal disease from early development to chronic kidney disease progression including renal fibrosis, diabetic nephropathy and the translational potential of identifying new biomarkersandtreatmentsforthepreventionandtherapyofchronic kidney disease and end-stage kidney disease. Keywords Epigenetics . Kidney . Chronic kidney disease . Diabetes . Epigenetic therapy

Introduction The concept of epigenetics was introduced by the developmental biologist Conrad H. Waddington in 1942 and describes

* Nicola Wanner [email protected] * Wibke Bechtel-Walz [email protected] 1

Department of Medicine IV, Faculty of Medicine, University of Freiburg, Freiburg, Germany

2

Center for Systems Biology (ZBSA), Albert-Ludwigs-University, Freiburg, Germany

3

Renal Division, University Hospital Freiburg, Breisacher Strasse 66, 79106 Freiburg, Germany

a mode of inheritance of traits not encoded in the DNA sequence (Waddington 2012). The information bestowed via epigenetic modifications can be influenced by the environment via the integration of data from the outside and the storage of such data in an epigenetic memory. Transgenerational inheritance of epigenetic traits has been described in plants (Hauser et al. 2011) and is a potentially adaptive mode of inheritance. In mammals, the occurrence of true transgenerational inheritance is still debated because of the erasure of epigenetic marks in the germline and preimplantation embryo, with the exception of imprinted genes and repetitive elements (Benyshek et al. 2006; Daxinger and Whitelaw 2012; Reik et al. 2001). However, intergenerational inheritance, such as in utero exposure or the influence of male sperm, has been described (Radford et al. 2014; Wei et al. 2014). Whereas the epigenetic profile of a cell mirrors its origin and can be used to distinguish between various cell types, some alleles change their epigenetic profile upon environmental cues. These alleles are termed metastable epialleles and cause the mosaicism of gene expression (Rakyan et al. 2002). The epigenetic profile has also been shown to be influenced by age or disease, resulting in consistent changes in DNA methylation or histone marks (Horvath 2013). A special form of epigenetic regulation, namely genomic imprinting, epigenetically silences one copy of a gene dependent on the sex of the parent (Li et al. 1993). In mammals, more than 70 imprinted genes have been identified with many of them being growth-factor-related, such as insulin-like growth factor 2 (IGF2); this discovery has lead to the postulation of the so-called Bconflict hypothesis^, in which the maternal copies try to restrict fetal growth, whereas the paternal alleles try to enhance it (Moore and Haig 1991). On the downside, inheritance of both copies from one parent leads to uniparental disomy with neurobehavioral symptoms, whereas that loss of imprinting can lead to a variety of cancers (Ferguson-Smith et al. 1990).

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Of the noncoding human DNA, more than 45% consists in transposable genetic elements, comprising retrotransposons, such as LINEs (long interspersed nuclear elements) and SINEs (short interspersed nuclear elements) and LTR (long terminal repeat) transposons and DNA transposons. These transposable elements undergo stable epigenetic silencing; however, their role in disease is unclear (Lander et al. 2001). DNA methylation In the mammalian genome, 60–90% of CpG (cytosine-phosphate-guanine) sites are methylated resulting in 5-methylcytosine (5mC; Ehrlich et al. 1982). In contrast to single CpGs, CpG islands are mostly unmethylated and often occur in promoters of housekeeping genes and actively transcribed genes. DNA methylation is a powerful means for transcriptional silencing and is involved in imprinting and Xchromosome inactivation and in the silencing of transposable elements. Maintenance methylation has been shown to be executed by DNA methyltransferase 1 (DNMT1), which can reestablish methylation patterns on newly synthesized strands via preferential methylation of hemi-methylated DNA and thus counteracts the dilutive effect of cell proliferation (Bestor 1992; Pradhan et al. 1999). On the other hand, de novo methylation can be achieved by DNMT3A and DNMT3B, which potentially change methylation patters on the DNA (Hsieh 1999; Okano et al. 1998, 1999). Opponents of the DNMTs are Ten-Eleven Translocation (TET) proteins, which reduce 5mC to 5-hydroxy-methyl-cytosine (5hmC; Tahiliani et al. 2009). The mechanism with which DNA methylation is thought to induce transcriptional silencing is the steric denial of access of proteins to the DNA because of a changed chromatin conformation. Additionally, proteins can be attracted to methylated DNA, such as Methyl-CpG binding domain (MBD) proteins and further mediate the epigenetic repression of transcription. Polycomb group proteins Another mechanism to establish epigenetic silencing involves the Polycomb group (PcG) of proteins originally discovered in Drosophila in which they silence Hox genes (Beuchle et al. 2001). Polycomb proteins form two major repressive complexes, containing different PcG proteins. Whereas Polycomb repressive complex (PRC) 1 containing Ring1 has E3 ligase activity and catalyzes mono-ubiquitinylated H2Aub1, PRC2 containing the EED-EZH2 subunits methylates histone H3 at Lysin 27 (H3K27me1-3) leading to transcriptional silencing (Kirmizis et al. 2004; de Napoles et al. 2004; Kuzmichev et al. 2004). Thus, by recruiting PRC2 to CpG islands, Polycomb group proteins synergize with the DNA methylation machinery (Mendenhall et al. 2010; Riising et al. 2014). By inducing the compaction of chromatin, PRC1 furthermore contributes to X-

chromosome inactivation in mammals (de Napoles et al. 2004; Francis et al. 2004; Hernandez-Munoz et al. 2005). Histone code Histone modifications have been detected at over 60 different sites, including acetylation, lysine and arginine methylation, phosphorylation, ubiquitylation and sumoylation, all of which are able to regulate gene transcription (Jenuwein and Allis 2001). Histone modifiers are divided into reader enzymes, such as histone acetylases and methyltransferases, eraser enzymes, such as deacetylases and demethylases and reader proteins, which do not change the epigenetic code but play a crucial role in reading, stabilizing integrating information and in recruiting an effector protein to a loci (Goldberg et al. 2007). Histone marks further play a role in chromosome condensation and higher order chromatin structure, dividing the chromatin into silent heterochromatin and active euchromatin. Active promoters have been described as being decorated with H3K4me3 and H3K27ac marks, active enhancers with H3K4me1 and H3K27ac marks and silenced promoters and enhancers with H3K27me3 marks (Shlyueva et al. 2014). Noncoding RNA Noncoding RNA acts as another regulator of cotranscriptional epigenetic silencing. Divided into small (<200 nucleotides) and long (>200 nucleotides) noncoding RNA, both classes influence gene expression but via different mechanisms. Long noncoding RNAs (lncRNA) influence gene expression and epigenetic mechanisms on the chromatin level (Mercer and Mattick 2013). lncRNA HOTAIR from the HOXC locus can alter the expression of the HOXD locus by interacting with PRC2 to induce H3K27me3 silencing (Rinn et al. 2007). Short noncoding RNA can be divided into three classes, namely microRNA (miRNA), small interfering RNA (siRNA) and piwi-interacting RNA (piRNA). Whereas miRNAs are 21–25 nucleotides long and can cleave mRNA with the help of the Dicer-containing RNA-induced silencing complex, siRNAs play a role in heterochromatin formation and chromosome condensation via the RNA-induced transcriptional silencing complex (Carthew and Sontheimer 2009). Similarly, the slightly longer 26- to 31-nucleotide piRNAs induce epigenetic and posttranscriptional silencing of transposons and can be transmitted maternally (Brennecke et al. 2008).

Epigenetics in prenatal renal reprogramming The nephron number is a major determinant of long-term renal function. A low nephron number has long been correlated with a heightened risk of developing hypertension and

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cardiovascular and chronic kidney disease (CKD; Brenner et al. 1988). In addition to genetic components, in utero environmental conditions have been shown to influence kidney size and nephron number. These conditions include prematurity, low birth weight, placental insufficiency, nutritional and metabolic alterations such as maternal diabetes (Gautier et al. 2015) and low maternal protein (Zeman 1968) and the consumption of toxins associated with smoking and drug abuse (dexamethasone, renotoxic drugs; MacLennan et al. 2004; Toledo-Rodriguez et al. 2012). However, the function and importance of epigenetic modifiers during physiological renal development is still mostly elusive. In the last few years, efforts have been made to unravel the role of various enzymes in the development of the embryonic kidney. Among them, transcription factor Pax2 has been shown to interact with Pax-transactivation domain interacting protein (PTIP), making it part of a Trithorax-like protein complex that establishes activating H3K4 methylation marks (Patel et al. 2007). On the other hand, Pax2 interaction with Grg4 makes it part of a Polycomb repressive complex inducing repressive H3K27me3 marks and thereby leading to gene silencing (Patel et al. 2012). Thus, Pax2 action is linked to epigenetic modifications in the developing kidney (Dressler and Patel 2015; Ranghini and Dressler 2015). Furthermore, histone deacetylases HDAC1 and HDAC2 bind to the promoters of major renal developmental genes (S. Chen et al. 2011). When the respective genes are deleted in the ureteric bud cell lineage, the kidneys are hypoplastic and show impaired cell proliferation and survival (S. Chen et al. 2015). Similarly, a mouse model of Dicer deletion in nephron progenitor cells or in the ureteric buds abrogate nephrogenesis indicating an important role of the miRNA machinery for kidney development (Nagalakshmi et al. 2011). As a mediator between environmental factors and their influence on renal development, the role of epigenetics has long been postulated. However, to this day, the connections are still vague and poorly characterized. As one example for the potential interplay between the environment and epigenetic regulation, a well-established connection has been made between in utero maternal smoking and differential DNA methylation rates. In comparison with newborns of nonsmoking mothers, the cord blood of babies whose mothers have smoked during pregnancy show CpG methylation near several genes, such as the xenobiotics-detoxifying genes arylhydrocarbon receptor repressor (AHRR) and a member of the cytochrome P450 superfamily CYP1A1 (Joubert et al. 2012) and reduced kidney volume (Taal et al. 2011). Similarly, fetal exposure to hyperglycemia in children from mothers with type I diabetes has been shown to influence global DNA methylation patterns, leading to a reduction in overall methylation rates in peripheral blood cells (Gautier et al. 2015). Although several animal models have shown that maternal high glucose exposure leads to reduced nephron endowment

(Amri et al. 1999; Kanwar et al. 2005; Tran et al. 2008), a similar connection has not yet been confirmed in humans in which hyperinsulinism experienced by the fetus can have opposite effects on growth (Mehta and Hussain 2003). Furthermore, changes in the DNA methylation of repetitive elements, such as LINE-1, have been shown in infants with low birthweight (Michels et al. 2011). This is also in agreement with animal models showing hypomethylation of the p53 promoter after intrauterine growth retardation leading to a decreased nephron number of 25% (Pham et al. 2003; Garro et al. 1991). Likewise, in utero alcohol exposure in rodent has been shown to lead to global hypomethylation (Garro et al. 1991). Other studies have confirmed the effect of ethanol on the methylation status on imprinted genes (Downing et al. 2011). As a substrate for DNA methyltransferases, increased methyl or cholin supplementation counteracts DNA changes and increases birthweight in some but not all studies (Downing et al. 2011; Bekdash et al. 2013; Garro et al. 1991). However, a direct effect on kidney development and function has not yet been shown and more work is needed to unravel the influence of environmental stressors on the epigenome of the kidney.

Epigenetics in diabetic nephropathy The number of patients with type I and type II diabetes mellitus are on a constant rise worldwide. About 30–40% of patients develop diabetic nephropathy (DN), which is the greatest risk factor for CKD and end-stage kidney disease (ESKD). In untreated diabetes, hyperglycemia leads to DN with persistent microalbuminuria, excessive extracellular matrix (ECM) accumulation and mesangial fibrosis, podocyte foot process effacement and apoptosis, thickened glomerular basement, glomerular sclerosis with Kimmelstiel-Wilson lesions and tubulointerstitial fibrosis (Kanwar et al. 2011; Reddy and Natarajan 2011). At the cellular level, hyperglycemia leads to advanced glycation end products activating nuclear factor kappa B (NF-κB) and transforming growth factor β1 (TGFβ1), which lead to inflammation. Furthermore, by crosslinking with the ECM and preventing its degradation, they lead to the accumulation of ECM and changes in the filtration barrier (Kanwar et al. 2011). DNA methylation in diabetic nephropathy Several studies have linked hyperglycemia and diabetic nephropathy to epigenetics. As a major transcriptional regulator, DNA methylation is the most widely studied epigenetic mechanism and has been shown to display many differentially methylated CpG sites between DN and control conditions (Brennan et al. 2010; Williams et al. 2008; Bell et al. 2010), e.g., the expression level of many genes, such as IGFBP-1,

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correlate with altered DNA methylation levels (Gu et al. 2014; X.H. Yang et al. 2016). Genes connected to DN in previous screens have been validated as being epigenetically regulated by DNA methylation (Bell et al. 2010; Sapienza et al. 2011). As part of the methyl cycle, methylenetetrahydrofolate reductase (MTHFR) was found to be demethylated in DN, indicating its possible role in the pathogenesis of DN (X.H. Yang et al. 2016). Interestingly, polymorphisms in this gene have also been implicated with an increased susceptibility of DN (Zhou et al. 2015). Furthermore, promoter demethylation of the connective tissue growth factor (CTGF) gene has been shown to be negatively correlated with the glomerular filtration rate (H. Zhang et al. 2014). Among mitochondrial genes, PMPCB (Peptidase, Mitochondrial Processing Beta Subunit), TSFM (Ts Translation Elongation Factor, Mitochondrial) and AUH (AU RNA Binding Methylglutaconyl-CoA Hydratase) have been found to be differentially methylated, indicating a role for mitochondrial function in diabetes (Swan et al. 2015). Furthermore, DNA hypermethylation has been shown to affect miRNA let-7a downregulation of UHRF1, an important interactor of Dnmt1 (Peng et al. 2015). However, although hyperglycemia has been shown to lead to demethylation in the rodent liver, global DNA methylation is not affected in the kidney (Williams et al. 2008). DNA methylation in human peripheral blood mononuclear cells is also not affected by hyperglycemia; nevertheless, albuminuria is correlated with DNA methylation levels (Maghbooli et al. 2014). Furthermore, differentially methylated CpGs have been identified in subjects with pre-diabetes mellitus transitioning to type II diabetes mellitus over a period of 7 years (VanderJagt et al. 2015). These genes are involved in carbohydrate and lipid metabolism and in inflammation and include genes such as SLC22A12, TRPM6, AQP9, HP, AGXT and HYAL2. Thus, DNA methylation seems to be directly involved in the pathogenesis of DN and to functionally influence disease progression by regulating gene expression. Histone modification in diabetic nephropathy As another major mechanism of epigenetic regulation, the modification of histone tails can influence gene transcription and the progression of disease. Both transient and persistent hyperglycemia leads to an increase in H3K4me1 levels in endothelial cells, indicating a role for histone methyltransferase SET7 in the execution of the epigenetic changes; this has also been validated in other conditions (Brasacchio et al. 2009; J. Chen et al. 2014). Mesangial cell cultures treated with high glucose also show an increased expression of SET7 induced by TGFβ1 (G. Sun et al. 2010). Furthermore, oxidant stress via 12/15-lipoxygenase-derived oxidized lipids can increase the expression of SET7 and profibrotic genes in DN (Yuan et al. 2016). Of the histone deacetylases, HDAC2/4/5

have been shown to be upregulated in streptozotocin-induced DN, db/db mouse models and diabetic patient kidney biopsies (X. Wang et al. 2014). Whereas HDAC4 seems to lead to inflammation in podocytes via Stat1 deacetylation (X. Wang et al. 2014), HDAC2 has been shown to play a role in renal fibrosis (Noh et al. 2009). Likewise, it is activated under diabetic conditions via ROS and TGFβ1 and induces epithelialto-mesenchymal transition (EMT) and fibrosis while inactivating antifibrotic genes. HDAC9 has also been shown to be upregulated in tissues from diabetic mice and patients (Liu et al. 2016). The silencing of HDAC9 can decrease glomerulosclerosis, inflammation and podocyte injury. The pro- and anti-inflammatory involvement of histone acetyltransferases (HATs) and HDACs has also been determined in mesangial cell cultures (Yuan et al. 2013). A similar deacetylating effect is produced by Apelin-13, further supporting the role of HDACs in DN (H. Chen et al. 2014). Other mouse models exhibit an increase in H3K9 and H3K27 acetylation and in H4K4me2 and the phosphorylation of H3S10 (Sayyed et al. 2010). Furthermore, histone methyltransferase enzyme enhancer of zeste homolog 2 (EZH2), part of the Polycomb repressor complex 2, has been demonstrated to have a protective role for podocytes and kidneys under diabetic conditions by decreasing TXNIP and PAX6 expression and inhibiting podocyte injury and oxidative stress (Siddiqi et al. 2016). Whereas HDACs have been shown to play a role in the expression of profibrotic genes in DN, deacetylating agents such as Apelin-13 inhibit the inflammation and expression of TGFβ1 and NF-κB (H. Chen et al. 2014, 2015). Acting in a similar fashion, broad class I and II HDAC inhibitor Trichostatin A (TSA) decreases ECM accumulation and EMT (Noh et al. 2009). A reduction of fibrogenesis and the expression of α-SMA, collagen I, fibronectin, TGFβ1 and NF-κB has also been demonstrated by the HDAC inhibitor sodium butyrate (Khan and Jena 2014; Dong et al. 2017). Another HDAC inhibitor, valproate, has been shown to ameliorate diabetic podocyte and renal injury via the induction of autophagy and the decrease of NF-κB action and of diabetesinduced fibrosis and apoptosis (Khan et al. 2015a, 2015b; X.Y. Sun et al. 2016). Whereas NAD-dependent deacetylase sirtuin-1 (SIRT1) is downregulated under diabetic conditions, 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (TSG) can counteract SIRT1 decrease and inhibit TGFβ1 action (Li et al. 2010; Kume et al. 2013). Use of the AT1R blocker losartan is able to reverse pathologic and epigenetic changes of DN in diabetic db/db mice (Reddy et al. 2014). Finally, type I diabetes involving a histone acetyltransferase interacting with NF-κB, p300, could be prevented by using curcumin (Chiu et al. 2009; Y. Wang et al. 2015). All in all, histone modifications seem to play a crucial role in the inflammatory response under diabetic conditions. Inhibition of HDACs shows great promise in the amelioration and prevention of

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diabetes-induced fibrosis and inflammation. The targeting of other epigenetic regulators, such as histone methyltransferase SET7, deacetylase SIRT1, or histone acetyltransferase p300, might additionally counteract DN disease progression, indicating the great potential of epigenetic drugs in the treatment of DN.

miRNA in diabetic nephropathy In addition to DNA methylation and histone modifications, noncoding RNA, especially miRNA, has been shown to play a major regulatory role in the development of DN. The first miRNA to be discovered in the context of diabetic kidney disease, miR-192, has been reported to represent a key regulator of collagen formation (Kato et al. 2007). In human renal biopsies from patients with diabetic nephropathy, the TGFβ-mediated upregulation of miR-192 in proximal tubule cells has been linked to fibrosis and the reduction in estimated glomerular filtration rate (eGFR; Krupa et al. 2010). Inhibition of kidney miR192 is associated with decreased renal fibrosis and reduced proteinuria in a diabetic kidney disease mouse model (Putta et al. 2012; Deshpande et al. 2013). Studies have shown that TGFβ1 leads to the acetylation of Ets1 and dissociation from the miR192 promoter (Dong 2013; Kato et al. 2013b). Another miRNA, miR-21, is increased in DN and might represent a potential therapeutic target (X. Zhong et al. 2013; McClelland et al. 2015; J. Wang et al. 2013; J.Y. Wang et al. 2014a). Whereas Hyperoside is a potential antagonist of miR21, serum miR-21 might also be a biomarker for DN (J. Wang et al. 2016). Another miRNA induced by TGFβ is miR-200b/ c, which contributes to glomerular mesangial hypertrophy (Park et al. 2013), whereas miR-200a seems to be involved in the negative feedback regulation of TGFβ (B. Wang et al. 2011). Anti-inflammatory effects have also been attributed to miR-146a, whose absence in podocytes leads to the accelerated development of diabetic glomerulopathy (Bhatt et al. 2016; Lee et al. 2016). Similarly, miR-29a can prevent DN in mice and decreases podocyte dysfunction under hyperglycemic conditions (H.Y. Chen et al. 2014; Lin et al. 2014). In contrast, miR-29c promotes fibrogenesis and podocyte apoptosis in the glomeruli of db/db mice (Long et al. 2011). Further miRNAs with an anti-inflammatory effect include miR-451, which counteracts NF-κB action in DN (Z. Zhang et al. 2012; Y. Sun et al. 2016). The upregulation of miR-34c prevents podocyte apoptosis under hyperglycemic conditions, whereas the overexpression of miR-26a prevents collagen deposition (Liu et al. 2015; Koga et al. 2015). Both miRNAs are downregulated in models of DN. Furthermore, the expression of miR-126 is negatively correlated with DN and albuminuria in patients and represents a potential biomarker for DN (AlKafaji et al. 2016; Barutta et al. 2016). The same has been shown for miR-130b (Lv et al. 2015; Bai et al. 2016).

However, many more miRNAs have been to promote fibrosis and apoptosis, including miR-195 (S. Chen et al. 2011; Y.Q. Chen et al. 2012), miR-1207-5P (Alvarez et al. 2013), miR-377 (Q. Wang et al. 2008), miR-135a (He et al. 2014), miR-218 (H. Yang et al. 2016) and miR-34a (L. Zhang et al. 2014; X. Zhang et al. 2016). The detection and characterization of miRNA in healthy subjects and DN patients has opened the potential for urinary biomarkers identifying miRNA such as miR-145 and miR-192 as potential markers in urinary exosomes (Barutta et al. 2013; Jia et al. 2016). Urinary miRNA has also been profiled in order to find early markers for microalbuminuria in patients with DN (Argyropoulos et al. 2015). As a potential treatment option, Tongxinluo has been shown to regulate miR-21 (J.Y. Wang et al. 2014b). Furthermore, curcumin inhibits miR-124 and prevents podocyte damage (Li et al. 2013). Finally, Artresatan downregulates miR-199b-5p leading to an increase in klotho expression, thereby preventing renal tubular damage in DN (Kang and Xu 2016). Thus, several miRNAs have been implicated in the development of renal fibrosis and disease progression under diabetic conditions and represent novel potential targets for therapeutic interventions or urinary biomarkers of disease.

Epigenetics in CKD and renal fibrosis The increasing incidence of CKD has become a major public health concern affecting almost 10% of the world population (Eknoyan et al. 2004). Tubulointerstitial renal fibrosis is the common end point of almost all chronic and progressive kidney diseases inevitably leading to renal function deterioration, independently of primordial renal disease. The two main causes of CKD, diabetes mellitus and hypertension, account for 60% of the cases and represent a major risk factor for cardiovascular disease (Coffman 2011). Further causes determining CKD are glomerulonephritis, inherited diseases (such as polycystic kidney disease), malformations, lupus and other autoimmune diseases, obstructions, or repeated urinary infections. Tubulointerstitial renal fibrosis is characterized by ECM deposition representing a dynamic system that involves renal and infiltrating cell types that exhibit enormous plasticity or phenotypic variability (Boor et al. 2010). EMT of tubular epithelial cells determines the progression of fibrosis in the kidney. Tubular epithelial cells can acquire a mesenchymal phenotype and the ability to migrate into the adjacent interstitial renal parenchyma (Efstratiadis et al. 2009; Kalluri and Neilson 2003; Zeisberg and Kalluri 2004). A multifaceted system of epigenetic regulators and transcription factors controls aberrant gene expression in CKD. Proteinuria and hypoxia are important stimulators of EMT initiation. TGFβ-1/SMAD signaling is considered as the main modulator regulating molecular mechanisms of EMT. The appearance of familiar

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clustering and the variabilities of CKD in diverse ethnic groups allow the assumption of a genetically inherited risk for the development of CKD (Smyth et al. 2014); however, genetics have proven to be insufficient to explain the heterogeneity of the progression rates of chronic renal disease among individual patients. Genome- and epigenome-wide next-generation sequencing and whole transcriptome studies have suggested epigenetic modifications as reasonable underlying mechanisms involved in the way that the environment dynamically interacts with the genome and with the inherited susceptibility to CKD and thus alters an individual’s risk of disease (Smyth et al. 2014). Recent technical advances have provided insight into the epigenetic control of proinflammatory and pro-fibrotic gene expression mediated by DNA methylation, histone modifications, changes in chromosome conformation, long non-coding RNAs and/or microRNAs, all of which can interact with one another, adding further complexity as to their involvement in gene regulation. So far, the analysis of epigenetic data from human material is puzzling, not only because of the lack of a complete epigenome reference but also because the epigenome is celltype-specific and because epigenetic heterogeneity within an organ still represents a diagnostic challenge. To date, epigenome-wide screening studies are still difficult to interpret and knowledge about the impact of epigenetics on the progression of CKD is only just evolving. DNA methylation in CKD and renal fibrosis Several experimental studies support the involvement of DNAme in renal fibrosis and CKD. Upregulation of the maintenance DNA methyltransferase 1 (Dnmt1), DNAme and transcriptional silencing have been linked to fibroblast activation and EMT in kidney fibrosis (Bechtel et al. 2010). Renal tubulointerstitial matrix deposition and overall reduced aberrant promoter methylation have also been demonstrated to be significantly reduced in Dnmt1 heterozygous knockout mice in experimental fibrosis supporting the impact of Dnmt1 on the progression of kidney fibrosis (Bechtel et al. 2010). In a study based on a genome-wide promoter methylation array, Wilms’ tumor 1 (WT1), a developmental master regulator that can also act as a tumor suppressor or oncoprotein, has been shown to influence the DNA methylation of gene promoters by transcriptionally regulating de novo DNA methyltransferase 3A (Dnmt3a; Szemes et al. 2013). Depletion of WT1 leads to the reactivation of gene expression from methylated promoters, e.g., the pro-fibrotic cytokine TGFβ2, the key modulator of EMT (Szemes et al. 2013). In the fibrosis of several organs, the TGFβ-isoform TGFβ1 has been shown to be upregulated and to inhibit the expression of the rasGAP-like protein 1 (RASAL1), a negative regulator of Ras signaling, by promoting DNAme at its promoter via DNMT1, leading to increased Ras-GTP signaling and increased fibroblast

activation and kidney fibrosis (Bechtel et al. 2010; Tao et al. 2011; Xu et al. 2015). In experimental renal disease models, hypermethylation of the Rasal1 promoter resulting in the reduced transcription of the gene has been linked to acute kidney injury and chronic progressive fibrosis (Bechtel et al. 2010). The epigenetically regulated transcriptional repression of Rasal1 has also been reported to cause fibroblast activation leading to liver fibrosis, suggesting a mutual role for Rasal1 in tissue fibrosis (Tao et al. 2011). One of the major recent discoveries in epigenetic research has shown that DNA methylation, similar to chromatin modifications, is potentially reversible by TET enzymes (Ito et al. 2010; Tahiliani et al. 2009). Aberrant DNAme of the Rasal1 promoter can be reversed by Tet3-mediated hydroxymethylation in cell culture, kidney biopsies and a mouse model, suggesting a potential novel therapy for CKD (Tampe et al. 2014). A recent extensive study determined that treatment with NAD-dependent deacetylase sirtuin-1 (Sirt1) improves albuminuria in proximal-tubule– specific Sirt1-deficient mice, via epigenetic mechanisms, including DNAme, by transcriptional repression of the tightjunction protein Claudin-1 in podocytes (Hasegawa et al. 2013) emphasizing the importance of intercellular communication between tubular cells and podocytes. Transcriptional repression of the transcription factor Kruppel-like factor 4 (KLF4) in podocytes has been associated with increased DNAme at the nephrin (Nphs1) promoter leading to podocyte apoptosis and proteinuria in animal models of renal disease, whereas KLF4 overexpression has renoprotective effects (Hayashi et al. 2014). Several recent studies have provided the first clinical evidence of an association between DNAme variations and fibrosis in human CKD by using relevant kidney samples. They represent an important step in CKD epigenome research (Beckerman et al. 2014; McCaughan et al. 2012; McKnight et al. 2014). Population-based studies for DNA methylation have been facilitated by the development of techniques to isolate sufficient numbers of specific renal cells (Ko et al. 2013) and by commercial arrays that now permit single CpG site resolution and epigenome-wide association studies (EWASs) for CKD at reasonable rates. A comprehensive genome-wide quantitative study evaluating DNA methylation for association with CKD revealed significant modifications at CpG islands of several genes including CUX1, ELMO1, FKBP5, INHBA-AS1, PTPRN2 and PRKAG2 (Smyth et al. 2014). Further investigation established that several of these genes are differentially methylated in kidney tissue and gene expression data support a functional role for the differential methylation in ELMO1 and PRKAG2 genes. Notably, the Protein Kinase AMPActivated Non-Catalytic Subunit Gamma 2, PRKAG2, has also been associated with CKD by genomic DNA singlenucleotide polymorphism studies (Kottgen et al. 2010). In the Chronic Renal Insufficiency (CRIC) Study comparing genomic DNA from participants having a fast rate of eGFR

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decline with patients showing improved GFR during followup, correlations have been found between the rate of loss of renal function and changes in DNAme at CpG islands of genes involved in EMT and fibrosis (e.g., NPHP4, IQSEC1 and TCF3) or oxidative stress and inflammation (e.g., NOS3, NFKBIL2, CLU, NFKBIB, TGFB3 and TGFBI) (Wing et al. 2014). DNAme profiling of tubuli from patients with CKD has demonstrated significant changes in DNAme versus normal controls and shown predominant variations occurring at enhancers that have also been correlated with the increased expression of key fibrotic genes of the TGFβ pathway (TGFBR3, SMAD3, SMAD6; Ko et al. 2013). Hypermethylation of the β-glucuronidase Klotho promoter and reduced gene expression have been reported to be associated with renal disease (J. Chen et al. 2013; Doi et al. 2011). In a recent study of a mouse model of adenine-induced CKD and unilateral ureteral occlusion-induced fibrotic kidney, the anthraquinone compound Rhein was reported to reduce profibrotic protein expression and renal fibrosis attributable to Klotho promoter hypermethylation by alleviating aberrant DNA methylation (Q. Zhang et al. 2016, 2017). Together, these recent findings clearly demonstrate that DNAme variations at key regulatory regions can be decisive in the development and progression of CKD. Further studies are needed to analyze and ensure whether reversal of the DNAme variations seen in the early stages of the disease can ameliorate renal dysfunction and/or prevent further progression to ESRD. Pharmacological interference with irregular epigenetic modifications has been shown to decelerate renal fibrogenesis. Demethylating drugs removing aberrant promoter DNAme are currently being tested in a number of clinical trials, most of them in cancer therapies. Administration of the demethylation agent 5-azacytidine ameliorates tubulointerstitial fibrosis in experimental renal fibrosis (Bechtel et al. 2010; C.Y. Sun et al. 2012). However, since 5-azacytidine and its derivate 5-aza-2deoxycytidine are randomly incorporated into the DNA affecting the methylation of not only atypically but also regularly methylated genes, these drugs produce severe side effects. Modification of the epigenome is a general process and most pharmacological agents available so far are cytotoxic and the long-term effects of genome-wide demethylation are insufficiently investigated. One of the most exhilarating recent advances in the epigenetic field is the discovery of TET-mediated hydroxymethylation as an endogenous mechanism of active DNA demethylation for the restoration of gene expression in stemness and pluripotency (Ito et al. 2010; Tahiliani et al. 2009). In the context of CKD, administration of bone morphogenic protein 7 (BMP7) has been linked to the Tet3-mediated reversal of pathologic hypermethylation at the Rasal1 promoter and the attenuation of experimental kidney fibrosis,

suggesting a new mechanism and potential therapeutic target for reversing kidney fibrosis (Tampe et al. 2014). Histone modifications in CKD and renal fibrosis Excessive expression of inflammatory and fibrotic genes has been linked to aberrant histone modifications in CKD (Reddy and Natarajan 2015). Histone acetylation in CKD and renal fibrosis The specific functions of HATs and HDACs in CKD progression are only incompletely understood (Hsing et al. 2012; Marumo et al. 2008, 2010; Yoshikawa et al. 2007). A recent study showed that the histone acetyltransferase p300/CBP-associated factor (PCAF) regulates inflammatory molecules (such as ICAM-1, VCAM-1 and MCP-1) through H3K18ac and causes the development of renal injury (Huang et al. 2015). In agreement with these findings, the pro-inflammatory cytokine TGFβ has been shown to promote p300 recruitment resulting in the activation of H3K9/14Ac at promoters of fibrotic genes, e.g., at Smad2/3, leading to increased fibrosis (Yuan et al. 2013). Another study demonstrated that TGFβ induces the acetylation of chromatin and of the transcription factor Ets-1 via Smads to enable the downregulation of miR-192, an important pro-fibrotic microRNA in mesangial cells in diabetic nephropathy (Kato et al. 2013b). In injured kidneys, decreased histone acetylation is associated with an upregulation of HDAC1, 2, 5 and 6 (Marumo et al. 2010), of which HDAC2 and 5 have been shown to facilitate the transcriptional suppression of reno-protective BMP7 (Hsing et al. 2012; Marumo et al. 2008; Yoshikawa et al. 2007). Histone methylation in CKD and renal fibrosis The principal regulatory transcription factor hypoxia-inducible factor 1 (HIF1) that controls gene expression under hypoxic conditions (Kaelin and Ratcliffe 2008) has been shown to induce the expression of histone demethylases in mammalian cells (Beyer et al. 2008; Krieg et al. 2010) and to promote dynamic changes in chromatin conformation (Mimura et al. 2012). Chronic hypoxia in the renal tubular interstitium results in increased fibrosis in CKD (Mimura and Nangaku 2010; Mimura et al. 2013; Nangaku 2006; Tanaka et al. 2005, 2006). Histone methylation is a mutual histone modification that is well studied in the context of histone marks. Among others, transcriptional activity is associated with H3K4me3 and transcriptional suppression is linked with H3K27me3 (Vastenhouw and Schier 2012). Abnormal histone methylation in the TGFβ pathway has been established as an important mechanism of fibrosis in CKD (G. Sun et al. 2010). TGFβ induces activating histone modifications and inhibits repressive marks at profibrotic gene promoters (G. Sun et al. 2010). In addition, TGFβ is also responsible for the upregulation of the H3K4-methyltransferase SET7 increasing

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H3K4me1 at TGFβ-induced pro-fibrotic genes (G. Sun et al. 2010). Furthermore, excessive incorporation of the histone isoform H2A.Z into the histone core in kidney injury is linked to activating H3K4me3 at pro-fibrotic genes encoding, for example, TGFβ1 and Type I collagen (Thatcher and Gorovsky 1994; Zager and Johnson 2009). Conditional deletion of the pax transactivation domain-interacting protein (PTIP; an essential component of a histone H3 lysine 4 [H3K4] methyltransferase complex) in glomerular podocytes in mice leads to ultrastructural defects in podocytes and chronic kidney failure in mice thereby demonstrating that modifications in an epigenetic regulatory pathway can even alter the phenotypes of differentiated cells leading to CKD (Lefevre et al. 2010). Recent studies have suggested epigenetic control mechanisms for the basal and aldosterone-induced expression of the tubular epithelial sodium channel (ENaC) that regulates blood pressure via sodium reabsorption in the renal collecting duct (Kone 2013). Under basal conditions, the H3K79methyltransferase Disrupter of Telomere Silencing (Dot)1a regulates the ENaCα expression promoter by complex formation with Sirt1 and other key proteins at the ENaCα promoter (Yu et al. 2013; W. Zhang et al. 2006, 2007, 2013; D. Zhang et al. 2009a, 2009b). In addition to ENaC, Angiotensin I converting enzyme (ACE1) controls blood pressure by activating the renin–angiotensin system. In a model of spontaneously hypertensive rats (SHRs), hypertension is associated with the upregulation of ACE1 (Ace) by facilitating activating marks (H3KAc and H3K4me) and decreasing repressive marks (H3K9me2) at the Ace promoter (Lee et al. 2012). Moreover, epigenetic mechanisms have been connected to ACE upregulation in the hypertensive offspring of mothers exposed to a low protein diet (Bogdarina et al. 2007) suggesting the need for more studies of epigenetics in hypertension and its potential heritability (Liang et al. 2013). Many studies have demonstrated a role of histone (de)acetylation in the progression of renal fibrosis. In a hypertensive nephrosclerosis animal model of Dahl salt-sensitive rats, the administration of curcumin, a histone acetyltransferase inhibitor, has been associated with the reduced acetylation of H3K9 and interleukin-6 gene expression, suggesting that curcumin ameliorates nephrosclerosis via the suppression of histone acetylation, independently of hypertension (Muta et al. 2016). In recent years, several studies have investigated the potentially favorable effects of HDAC inhibitors in animal models of acute kidney injury (e.g., in a zebrafish model; Cianciolo Cosentino et al. 2013) and CKD (Advani et al. 2011; Gilbert et al. 2011; Kosanam et al. 2014; Lin et al. 2014; Van Beneden et al. 2011, 2013; Yoshikawa et al. 2007). In comparison with de-methylating drugs and removing aberrant DNAme, HDAC inhibitors show fewer side effects attributable to the more specific disruption of HDAC substrates; however, robust data are not as yet available. HDAC inhibition through treatment with the HDAC inhibitor Vorinostat, which has been FDA approved for the treatment of cutaneous T cell

lymphoma (CTCL), has renoprotective qualities (Advani et al. 2011; Y. Zhong et al. 2013). In a recent study, the application of Vorinostat normalized EGFR-mediated signaling and attenuated experimental renal fibrosis (Gilbert et al. 2011). Renoprotection and the improvement of experimental fibrosis by controlling epidermal growth factor receptor (EGFR) endocytic trafficking and degradation in renal epithelial cells can also be achieved by the administration of Tubacin, a specific HDAC6 inhibitor, suggesting a key role for HDAC6 in the progression of CKD (Liu et al. 2012). Histone H3 phosphorylation causes the G2/M phase arrest of tubular epithelial cells preventing their capability for regeneration and proliferation in CKD (L. Yang et al. 2010). The Class-Iselective HDAC inhibitor MS-275 inhibits TGFβ1 signaling and rescues G2/M cell cycle arrest of tubular epithelial cells thereby alleviating renalfibrosis (Liu et al. 2013; Marumo etal. 2010; Noh et al. 2009; Pang et al. 2009; Van Beneden et al. 2013). Another study revealed that phenylbutyrate and valproic acid, class I and II inhibitors, ameliorate streptozotocin (STZ)-induced diabetic nephropathy, doxorubicine-induced nephropathy and experimental kidney fibrosis (Noh et al. 2009; Qi et al. 2011; Van Beneden et al. 2013). Moreover, the administration of the HDAC Class I and II inhibitor trichostatin A or of the α2-adrenoreceptor-agonist dexmedetomidine attenuates intra-renal inflammation and tubulointerstitial fibrosis in various mouse models of experimental kidney fibrosis through increasing BMP7 expression and inhibiting EMT via HDAC2 and/or HDAC5 inhibition (Bieliauskas and Pflum 2008; Hsing et al. 2012; Marumo et al. 2008, 2010; Yoshikawa et al. 2007). As the mechanism of action and selectivity of HDAC inhibitors and other epigenetic modifications including histone KMe and DNAme are not fully clear, future studies will have to elucidate their efficiency in the treatment of renal disease and to develop novel screening approaches to discover additional epigenetic modulators specific to renal diseases. miRNA in CKD and renal fibrosis Among all epigenetic mechanisms, RNA interference through microRNAs (miRs) is the most dynamic. MiRs have a key role in a variety of biological processes that influence renal development and homeostasis and the progression of kidney fibrosis (Brennan et al. 2013; Kantharidis et al. 2011; Kato 2013; Kato et al. 2013a; Kato and Natarajan 2012, 2014; Sato et al. 2011; Schena et al. 2014; Tampe and Zeisberg 2014; B. Wang and Ricardo 2014). Downregulation of Dicer, the essential enzyme in the production of miRs, in mouse podocytes leads to renal failure and death (Matsuda et al. 2000; Nagalakshmi et al. 2011; Patel and Noureddine 2012; Shi et al. 2008). For CKD, TGFβ-regulated miR-21, miR-200 and miR-29 have been reported to play a role in renal fibrosis (Patel and Noureddine 2012). miR-21 amplifies TGFβ signaling by targeting peroxisome proliferator-activated receptor-α (PPARα; Chau et al. 2012) and promotes fibrosis. Conversely,

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target mRNAs involved in inflammatory response, cell-cell interaction, apoptosis and intra-cellular signaling, clinical parameters and histological damage indices (Rudnicki et al. 2016). As a result, miRNAs exemplify novel biomarkers and potential therapeutic targets for CKD.

miR-200 and miR-29 reduce fibrosis by inhibiting EMT (Keller et al. 2012; Qin et al. 2011). In renal tubular cells, miR-205 expression modulates cell susceptibility to both oxidative and endoplasmic reticulum stresses via the suppression of the Egl-9 family hypoxia inducible factor 2 (EGLN2) and the subsequent decrease in intracellular ROS (Muratsu-Ikeda et al. 2012). Renal epithelial miR-205 expression has also been linked to the regulation of apoptosis in renal cells by targeting the CKLF Like MARVEL Transmembrane Domain Containing 4 (CMTM4), which is involved in cell proliferation and apoptosis (H. Zhang et al. 2015) and to the extent of disease severity in a mouse model of congenital obstructive nephropathy regulating urothelial differentiation (Wilhide et al. 2016). Recently, miR-146a has been described to function as a key mediator of the renal tubular response to ischemia-reperfusion injury preventing the development of inflammation and fibrosis by controlling interleukin-1 receptor-associated kinase 1 and C-X-C motif ligand 8 (CXCL8)/ CXCL1 expression in injured tubular cells (Amrouche et al. 2016). miRNA and mRNA expression profiling in renal biopsy sections has discovered that miR-30d, miR-140-3p, miR532-3p, miR-194, miR-190, miR-204 and miR-206 are downregulated in progressive cases correlated with upregulated 29

DNAme, histone modifications and miRs influence inflammatory and pro-fibrotic genes to perpetuate renal disease progression and outclass genetic factors unable to explain sufficiently the heterogeneity of progression rates of individual patients. Basic scientific animal models, clinical studies and the diagnostic potential to produce large amounts of epigenomic data suggest a critical role for epigenetic modifications in renal development and CKD progression (Fig. 1). The modifiable nature and reversibility of epigenetic marks by currently licensed drugs will aid the design of novel epigenetic drugs having promising clinical applications with regard to CKD. More than ever, an urgent need exists for the discovery of new diagnostics, biomarkers and therapies in clinical nephrology to tackle the central problem of the virtual

Fig. 1 Epigenetic influence on renal disease. Increasing evidence points to the involvement of epigenetic regulation in the development of renal disease in which the underlying causes, such as diabetes and hypertension, or stressors, such as inflammation and oxidative stress, can induce epigenetic changes including DNA methylation, histone

modifications, or changes in miRNA expression. The effect of epigenetic modifications on gene expression and cellular function plays a key role in the development of kidney disease (Me methyl group, Ac acetyl group, Ub ubiquitin group, CKD chronic kidney disease, ESKD end-stage kidney disease)

Future perspectives for diagnostics

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unopposed progression of renal diseases. The latest developments in oncologic research provide hope that the epigenetic therapies that are currently being used in cancer treatment, such as the correction of CpG methylation through the administration of de-methylating agents or the targeting of histonemodifying enzymes (Dawson and Kouzarides 2012; Fenaux et al. 2009; Jones 2012), will be transferred into clinical usage in patients with CKD. However, the low specificity and/or selectivity of these drugs and their adverse effects have still to be overcome. Interestingly, conventional therapies combined with or without (new) epigenetic drugs have secured renal protection and prevent the appearance or progression of CKD. A retrospective study of patients with hypertensive

nephrosclerosis suggests low-dose therapy with the directacting smooth muscle relaxant Dihydralazine to reduce CKD progression by TET methylcytosine dioxygenase 3/thymine DNA glycosylase (Tet3/Tdg)-mediated promoter demethylation of the ras inhibitor RASAL1 (Tampe et al. 2015). In a mouse model of ischemia-reperfusion injury, the administration of low-dose hydralazine effectively attenuated progression into renal fibrosis and preserved excretory renal function independently of its blood pressure–lowering effects (Tampe et al. 2016). In a mouse model, the combination of the ACE inhibitor Benazepril and the HDAC inhibitor Vorinostat significantly reduced the progression of HIV-induced nephropathy compared with each drug alone (Y. Zhong et al. 2013).

Fig. 2 Epigenetic regulation and therapy of kidney disease. The main causes of CKD are diabetes, hypertension and glomerulonephritis. As a common downstream pathway, these primary diseases lead to inflammation and extracellular matrix (ECM) accumulation resulting in tubulointerstitial renal fibrosis or diabetic nephropathy and finally in chronic kidney disease (CKD) and end-stage kidney disease (ESRD). Many epigenetic modifiers have been shown to play a role in the development of renal disease and several inhibitors (red) have successfully been shown to ameliorate inflammation and fibrosis in disease models

(ROS reactive oxygen species, AGE advanced glycation end products, TSA Trichostatin A, TGFb1 transforming growth factor beta 1, SIRT1 Sirtuin 1, TSG tetrahydroxystilbene glucoside, HDAC histone deacetylase, MS-275 Entinostat, PCAF p300/CBP-associated factor, RASAL1 RAS Protein Activator Like 1, NaBu sodium butyrate, 5-Aza 5-azacytidine, DAC decitabine, KLF4 krüppel-like factor 4, EGFR epidermal growth factor receptor, EMT epithelial-to-mesenchymal transition, GBM glomerular basement membrane, miRNA microRNA, SET7 histone methyltransferase)

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Likewise, we need to identify biological markers that help to identify patients who are at a higher risk of developing CKD than others, so that targeted therapies can be used to delay the onset or progression of CKD. At the same time, patients at low risk for CKD can avoid exposure to unnecessary drug treatment. Next-generation sequencing can improve the refinement of epigenetic risk factors and lead to the identification of a clinically useful epigenetic risk profile (Atzler et al. 2014; Chasman et al. 2012; Siggens and Ekwall 2014). However, personalized medicine requires robust tools to identify genetic and epigenetic risk factors associated with CKD in order to enhance risk estimation for CKD by integrating clinical, molecular and environmental data. Personalization and individual treatment options for patients at high risk of developing CKD will enhance patient care, facilitate the optimization of therapies and potentially reduce the healthcare costs of CKD. The use of epigenetic marks in renal tissue associated with CKD as a diagnostic tool for individual risk estimation of renal disease progression might increase our understanding of the biological mechanisms underlying CKD. However, the cellular heterogeneity of the kidney and the invasive nature of the collection of a kidney biopsy might limit the usefulness of this technique for routine clinical use. Body fluids can be sampled in a minimally invasive manner and represent easily accessible reservoirs for epigenetic biomarkers (whether by using whole blood or in silico techniques to adjust for the different cell types) for detecting kidney complications and predicting patients at high risk of developing CKD. Their ready accessibility in blood and urine makes miRNAs attractive biomarkers or targets for therapeutic intervention (Ramachandran et al. 2013). Potential epigenetic biomarkers for CKD progression have also been identified in site-specific DNA methylation levels in DNA extracted from the saliva of diabetes patients with ESRD compared with diabetes patients without nephropathy (Sapienza et al. 2011). Recently, circulating methylated DNA promoter fragments have been correlated with intrarenal levels of methylated promoter CpG islands of respective genes and the degree of renal fibrosis, suggesting the possibility of non-invasively analyzing intrarenal CpG island methylation and linking it to the degree of fibrosis (Tampe et al. 2015). An understanding of the epigenetic modifications in CKD will facilitate the prediction of the extent of the disease and help in the implementation of targeted therapies to prevent the progression of CKD (Fig. 2). Acknowledgments The authors gratefully acknowledge the support of their work by a Marie Curie EU grant (CIG 293568; to W.B.W.), the Margarete von Wrangell Habilitationsprogramm, Ministerium für Wissenschaft Baden-Württemberg (to W.B.W.) and the MathildeWagner-Habilitationspreis (to W.B.W.). This study was also supported by the German Research Foundation (DFG) within the CRC 1140 and CRC 992. The authors thank all members of their laboratory for their support and helpful discussions and apologize to those colleagues whose work has not been cited because of length restrictions.

Compliance with ethical standards Disclosures The authors declare that they have no conflicts of interest.

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