Nephrology Dialysis Transplantation

NDT Advance Access originally published online on February 28, 2008
Nephrology Dialysis Transplantation 2008 23(5):1493-1496; doi:10.1093/ndt/gfn056

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Epigenetics—a helpful tool to better understand processes in clinical nephrology?

Peter Stenvinkel¹ and Tomas J. Ekström²

¹ Division of Renal Medicine, Department of Clinical Science, Intervention and Technology ² Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden

Correspondence and offprint requests to: Peter Stenvinkel, Department of Renal Medicine, K56 Karolinska University Hospital at Huddinge, 141-86 Stockholm, Sweden. Tel: +46-8-58582532; Fax: +46-8-7114742; E-mail: peter.stenvinkel{at}ki.se

Keywords: epigenetics; chronic kidney disease; genetics; homocysteine; inflammation

	Epigenetic processes control the packaging and function of the human genome

Top Epigenetic processes control the... How can epigenetics be... Factors affecting the epigenome... Impact of epigenetics on... Can epigenetics be manipulated? Conclusion References

 
Epigenetics (literally in addition to the genetic sequence) is a novel discipline that has languished in the shadow of its genomic big brother that has attracted little interest among nephrologists. By tradition, phenotypic variations are divided into a genetic and an environmental component (Figure 1). There is no doubt that variations within the genome may have an impact on the phenotype in chronic kidney disease (CKD) [1]. However, as epigenetic mechanisms due to environmental factors are also critical for normal functioning of the genome [2], the associations between the unphysiological uraemic environment and the epigenotype should be of interest to study in this patient group. Indeed, as the epigenotype is transmitted to daughter cells, and epigenetic changes may endure in subsequent cell generations, this discipline could bring a new perspective to the study of all physiological processes.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 The phenotype is not only determined by genotype (DNA sequence) and the environment (external impact), but also the epigenotype, which is influenced by the combinations of these. SNPs; single nucleotide polymorphism.

 
The epigenetic state, and thus the interpretation of the genome is based on the recognition by proteins involved in transcription of specific genomic loci. Chromatin consists of DNA wrapped around cores of histones. The specific enzymatic modifications of DNA and histones are specific for a given locus in a given cell type at a given time and under defined outer influences. The unique structure of chromatin formed at specific loci provides the specificity of the genome to transcription required to acquire and maintain cell-specific phenotypes. The modifications that can occur in eukaryotes include methylation at the 5-carbon position of cytosines in CG (CpG) dinucleotide sequences. The mammalian epigenome denotes properties of the genome that are not explained by the primary DNA sequence but rather by the results of (a) modifications of DNA due to heritable (but reversible) changes in cytosine methylation; (b) modifications of histones (chromatin modifications), such as acetylation, methylation, phophorylation and ubiquitination; (c) energy-dependent chromatin remodelling and (d) RNA-based silencing (Figure 1). Epigenetic studies of various species have shown that epigenetic regulation is critical for the normal function of the genome. However, if they occur improperly they may cause dysregulations that promote cancer [3]. In addition, several disease processes commonly observed in CKD, such as ageing [4], diabetes mellitus [5], autoimmune disease [6] and vascular disease [7], have been associated with epigenetic changes. Moreover, as a recent study in rats suggested a link between fetal insults and epigenetic modification of gene expression and the development of hypertension in adult life [8], epigenetics may also play a role in the development of human hypertension.

Recently, several groundbreaking studies have suggested that environmentally induced epigenetic modifications can be inherited for generations. In fact, alterations in epigenetic marking of the genome may be a mechanism by which nutritional exposure in utero can influence gene expression and phenotype. Indeed, a recent study of female sheep showed that by restricting the availability of vitamin B12, folate and methionine from the periconceptional diet, widespread epigenetic modifications (associated with adiposity, insulin resistance and high blood pressure) were found in the offspring [9]. Interestingly, there seems to be sex-specific transgenerational responses to previous nutritional exposure also in humans [10]. As a Swedish study showed that diabetes mortality increased if the paternal grandfather was exposed to a surfeit of food during his slow growth prepubertal period [11], it seems like we do not only inherit our gene sequence but also the effects of our ancestors lifestyle.

Although monozygotic twins share a common genotype, several types of phenotypic discordance, as well as different susceptibility to disease, can be observed at later stages of their life. Indeed, discordance of monozygotic twins seems to be a hallmark of many non-Mendelian complex diseases. Interestingly, there have also been reports on a discordant evolution in monozygotic twins with renal disease [12]. As monozygous twins exhibit remarkable differences in the overall content and genomic distribution of 5-methylcytosine-containing DNA and histone acetylation [13], a different phenotype can, indeed, originate from the same genotype.

	How can epigenetics be studied?

Top Epigenetic processes control the... How can epigenetics be... Factors affecting the epigenome... Impact of epigenetics on... Can epigenetics be manipulated? Conclusion References

 
In contrast to genetic studies, the assessment of epigenetic modifications requires access to the tissue/cell type of interest. Unfortunately, this makes many epigenetic investigations in human studies virtually impossible. As an extreme case, the study of epigenetics in connection to human behaviour would require hippocampal biopsies. Therefore, for such studies, surrogate tissues and/or autopsy material have to be used. The assessment of DNA methylation can be performed on most biological materials, fresh as well as frozen, at the genome-wide global scale, or gene-specific. Several methods for global and gene-specific methylation analysis have been used [14]. The use of methylation-sensitive and -insensitive isoschizomers of restriction endonucleases forms the basis for several approaches. One enzyme is able to cut only unmethylated DNA, whereas the other cuts both methylated and unmethylated DNA. HpaII and MspI, which recognize CCGG sequences, are commonly used for studying DNA methylation. After DNA cleavage, the comparative methylation state can be determined in several different ways. Genome-wide CpG methylation has been assessed using the HpaII/MspI isoschizomers by applying several different methods, e.g. self-primed in situ labelling (SPRINS) [15], methylation-sensitive arbitrarily-primed polymerase chain reaction (AP-PCR) [16], non-methylated genomic sites coincidence cloning [17], differential methylation hybridization [18] and methylation target array [19]. A method based on methylation-sensitive restriction enzymes followed by quantification of the digested sites can be performed in large patient materials using the luminometric assay LUMA, which can be used as a quantitative rather than a qualitative method [13,20]. Other, rare cutting methylation-sensitive restriction enzymes can be used to investigate and localize DNA methylation in screening methods like restriction landmark genomic scanning [21]. Chromatographic assays like capillary electrophoresis, measuring the content of total methylcytosine, is another concept to achieve a picture of total genomic DNA methylation [22].

For site-specific information of DNA methylation, bisulphite-converted DNA is frequently used with a number of methods. This results in unmethylated cytosines being converted to uracil (and subsequently thymidine if PCR amplified), while methylated cytosines remain unchanged. This forms the basis for a few useful assays. A widely used method for site-specific methylation is methylation-specific PCR (MSP), in which bisulphite-converted DNA is subjected to PCR over a specified sequence [23]. One important feature of MSP is its very high sensitivity where a subset of cells in a population can be assessed for methylation changes in a specific locus. However, for the detailed analysis of DNA methylation, sequencing of bisulphite-converted DNA is the method of choice. Different sequencing platforms, including pyrosequencing [24], can provide methylation information of every cytosine in a sequence-specific way. High through-put technologies [25] using bisulphite-converted DNA provide genome-wide site-specific methylation information, but due to its high cost, bisulphite sequencing is currently mostly used for the interrogation of subsets of genes.

Depending on the questions asked, global and gene-specific DNA methylation analyses may both be useful when assessing pathological conditions. However, although global analysis is a blunt tool, it should be emphasized that (in analogy to the unspecific nature of fever in various diseases) aberrant global DNA methylation is a sign of an epigenetic dysregulation that may provide information of general disease states.

	Factors affecting the epigenome in the uraemic milieu

Top Epigenetic processes control the... How can epigenetics be... Factors affecting the epigenome... Impact of epigenetics on... Can epigenetics be manipulated? Conclusion References

 
Several features of uraemia, such as hyperhomocysteinaemia [26], dyslipidaemia [27], inflammation [28] and oxidative stress, may promote aberrant DNA methylation. The homocysteine precursor S-adenosylhomocysteine (SAH) is a competitive inhibitor of the main methyl group donor in DNA methylation reactions—S-adenosylmethionine (SAM) [29]. In patients with vascular disease, increased homocysteine and SAH concentrations were associated with DNA hypomethylation [30]. Thus, as plasma SAH levels are positively associated with lymphocyte DNA hypomethylation [31], elevation of homocysteine seems to have a negative effect on methylation reaction mediated via an increase in intracellular SAH. Accordingly, Ingrosso et al. [26] found signs of DNA hypomethylation in mononuclear cells in a selected group of 32 hyperhomocysteinaemic HD patients.

As persistent inflammation may contribute to the counterintuitive association between low homocysteine levels and outcome in CKD [32], it is of interest that inflammation may also promote aberrant DNA methylation [33]. Whereas one study showed that the inflammatory cytokine IL-6 might exert an impact on epigenetic changes in cells via regulation of a DNA methyltransferase gene [34], another study by the same group showed that IL-6 supports tumour suppressor gene promoter methylation [35]. In CKD 5 patients, aberrant DNA hypermethylation is associated with inflammation and poor outcome and in vitro IL-6 promotes DNA hypermethylation [28]. As a recent study of 42 dialysis patients with renal neoplasm showed evidence of DNA hypermethylation of various genes compared to renal neoplasm cases with normal renal function, it appears that CKD and/or the dialysis procedure per se promotes hypermethylation [36]. As folic acid affects global DNA methylation status in the genome and the regulation of imprinted genes [26], vitamin status is another factor that should be taken into account when studying the complex and context-sensitive interactions between aberrant DNA methylation and outcome in CKD. Moreover, as methylation status correlates with the transcriptional activity of a promotor, and CpG island methylation is associated with gene silencing [37], the effects of DNA hypermethylation on the expression of different genes in the uraemic milieu need further evaluation. Indeed, one study demonstrated that DNA hypermethylation causes silencing of erythropoietin expression [38].

	Impact of epigenetics on gene expression and telomere attrition

Top Epigenetic processes control the... How can epigenetics be... Factors affecting the epigenome... Impact of epigenetics on... Can epigenetics be manipulated? Conclusion References

 
Epigenetic mechanisms of gene regulation may be crucial determinants of cellular behaviour. DNA methylation changes may occur during atherogenesis and contribute to lesion development by alterations in specific gene expression. As Lund et al. [39] showed that changes in DNA methylation profiles (i.e. both hypo- and hypermethylation) are early markers of atherosclerosis in a mouse model, it could be speculated that changes in DNA methylation will lead to aberrant gene expression and the transition from a normal cellular phenotype to one that predisposes to vascular lesions. Indeed, inactivation of the oestrogen receptor alpha gene due to methylation has been demonstrated to play a role in atherogenesis and ageing of the vascular system [40]. As aberrant DNA methylation (by down-regulation of atherosclerosis-protective genes and/or up-regulation of atherosclerosis-susceptible genes) may contribute to atherosclerosis, epigenetic changes of DNA function should be of major interest to study in CKD.

Telomeres are nucleoprotein complexes protecting the chromosome ends that are involved in chromosome stability and repair. Telomere shortening (attrition) has been associated with an inflammatory phenotype and increased mortality in both non-renal [41] and CKD 5 [42] patients. In this context, it is of interest that telomere shortening to a critical length results in loss of histone and DNA methylation at mammalian telomeres, concomitant with increased histone acetylation [43]. Thus, the link between epigenetic status and telomere-length regulation may provide a new avenue for the understanding of the pro-atherogenic cellular senescent phenotype, a disease process characterized by telomere-length defects [44].

	Can epigenetics be manipulated?

Top Epigenetic processes control the... How can epigenetics be... Factors affecting the epigenome... Impact of epigenetics on... Can epigenetics be manipulated? Conclusion References

 
Epigenetics may not only uncover etiologic and pathogenic mechanisms, but also be a new concept that provides the basis for the development of future treatment strategies targeting the primary epigenetic causes of many diseases. In theory, epigenetic drugs could possess higher therapeutic potential and much lower rate of adverse effects in comparison to current treatments [45]. Currently, a collection of epigenetic drugs, such as DNA methylation inhibitors and histone deacetylase inhibitors, exist at various stages of development [45]. Although promising results have been reported, major concerns include their promiscuity (i.e. lack of target specificity) as well as their transient effects [45]. As interventions with folate in man [26], and genistein in mice [46], may reverse epigenetic DNA modification, there is a possibility of future nutritional interventions directed at modifying the epigenome in CKD.

	Conclusion

Top Epigenetic processes control the... How can epigenetics be... Factors affecting the epigenome... Impact of epigenetics on... Can epigenetics be manipulated? Conclusion References

 
Our understanding of the roles played by epigenetic modifications in CKD remains in its infancy. Thus, nephrologists may, in this novel discipline, find an ocean of information largely unexplored, which holds the potential to explain at least some of the influence of the uraemic environment on the pro-atherogenic phenotype that characterizes these patients. Not only should the associations of aberrant DNA methylation in relation to homocysteine, dyslipidaemia, vitamin deficiencies, oxidative stress and inflammation be better explored, but also the impact on the epigenome of different dialysis procedures and nutritional status. Further research is also needed to study the association between aberrant global DNA methylation, gene level methylation status and silencing of genes in the uraemic milieu. Finally, as epigenetic DNA modifications are potentially reversible, the effects of various epigenetic interventions on outcome in CKD should be studied.

	Acknowledgments

 
The Swedish Heart and Lung Foundation (P.S.), the Swedish Medical Research Council (P.S.) and the Swedish Cancer Foundation (T.J.E.) supported the present work. We thank Anders Alvestrand and Mohsen Karimi for constructive criticism.

Conflict of interest statement. None declared.

	References

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Received for publication: 25.10.07
Accepted in revised form: 23. 1.08

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This Article Extract FREE Full Text (PDF) All Versions of this Article: 23/5/1493    most recent gfn056v2 gfn056v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Stenvinkel, P. Articles by Ekström, T. J. Search for Related Content PubMed PubMed Citation Articles by Stenvinkel, P. Articles by Ekström, T. J. Social Bookmarking          What's this?

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