Epigenetics and epigenomics in diabetic kidney disease and metabolic memory

Abstract

The development and progression of diabetic kidney disease (DKD), a highly prevalent complication of diabetes mellitus, are influenced by both genetic and environmental factors. DKD is an important contributor to the morbidity of patients with diabetes mellitus, indicating a clear need for an improved understanding of disease aetiology to inform the development of more efficacious treatments. DKD is characterized by an accumulation of extracellular matrix, hypertrophy and fibrosis in kidney glomerular and tubular cells. Increasing evidence shows that genes associated with these features of DKD are regulated not only by classical signalling pathways but also by epigenetic mechanisms involving chromatin histone modifications, DNA methylation and non-coding RNAs. These mechanisms can respond to changes in the environment and, importantly, might mediate the persistent long-term expression of DKD-related genes and phenotypes induced by prior glycaemic exposure despite subsequent glycaemic control, a phenomenon called metabolic memory. Detection of epigenetic events during the early stages of DKD could be valuable for timely diagnosis and prompt treatment to prevent progression to end-stage renal disease. Identification of epigenetic signatures of DKD via epigenome-wide association studies might also inform precision medicine approaches. Here, we highlight the emerging role of epigenetics and epigenomics in DKD and the translational potential of candidate epigenetic factors and non-coding RNAs as biomarkers and drug targets for DKD.

Fig. 1.. Molecular mechanisms of diabetic kidney disease.

The pathophysiology of diabetic kidney disease (DKD) is complex and involves interactions between genetic factors, epigenetic factors, and the environment. High glucose levels in the context of diabetes mellitus, together with hypertension, and hyperlipidaemia induce the expression of growth factors, angiotensin II (AngII), cytokines, advanced glycation end products (AGEs), and produce oxidized lipids, mitochondrial dysfunction, and oxidative and endoplasmic reticulum (ER) stress. Growth factors, such as such as transforming growth factor β1 (TGF-β1), stimulate signal transduction pathways, including those involving MAPK and AKT kinases that activate downstream transcription factors, such as upstream stimulatory factors, SMADs, AP1 and the ER stress-related transcription factor, CHOP. They also affect epigenetic processes, including DNA methylation, histone modifications, and the expression of non-coding RNAs (microRNAs and long non-coding RNAs), resulting in altered chromatin accessibility and/or altered expression of target genes. Interactions between transcription and epigenetic factors lead to the persistent activation of signalling pathways, which might contribute to the phenomenon of metabolic memory, whereby exposure to high glucose levels leads to continued detrimental effects, even after glycaemic control has been established. Together, these conditions promote inflammation, fibrosis, and glomerular hypertrophy, which are hallmarks of DKD.

Fig. 2.. The effects of epigenetic modifications on chromatin structure.

Chromosomes are composed of DNA–protein complexes called chromatin. The basic subunit of chromatin is the nucleosome, which comprises an octamer of two copies of each of the core histone proteins H2A, H2B, H3 and H4, wrapped by 147 base pairs of chromosomal DNA. Condensed chromatin is generally characterized by promoter DNA methylation (Me) and histone Me depending on the site of modification on histone (for example, lysine 9 methylation (K9Me) and lysine 27 methylation (K27Me) on histone H3), resulting in repressed gene expression. Relaxed chromatin is marked by histone lysine acetylation (Ac) and methylation of lysine 4 on histone 3 (K4Me) in the promoter regions, which allows efficient gene expression. Histone lysine acetylation at the enhancer region also enhances gene expression by interacting with the promoter region through bromodomain-containing proteins (BRD) and other proteins. RNA methylation (Me) is a form of epigenetic regulation related to the translation and degradation of RNAs.

Fig. 3.. Epigenetic mechanisms of metabolic memory.

Hyperglycaemia alters histone modifications and DNA methylation status, resulting in a change in chromatin structure from condensed (marked by DNA methylation (Me), methylation of lysine 9 on histone 3 (K9Me), and methylation of lysine 27 on histone 3 (K27Me)), which is associated with repressed gene expression, to relaxed (marked by histone lysine acetylation (Ac) and methylation of lysine 4 on histone 3 (K4Me)) to induce the expression of pathogenic genes. Acetylation relaxes chromatin and enhances gene expression through various mechanisms, including by facilitating the release of transcriptional repressors from condensed chromatin. Once established, the chromatin status (including DNA methylation and histone modification status) is maintained, even after normoglycaemia has been established by administration of insulin or antidiabetic drugs, with persistent expression of pathogenic genes, in a phenomenon called ‘metabolic memory’. This epigenetic memory could potentially be erased using epigenetic drugs or by inducing locus-specific changes in histone modifications or DNA methylation through approaches such as gene editing directed by guide RNAs complementary to a specific locus.

Fig. 4.. Contribution of signalling circuits to persistent long non-coding RNA expression.

a ∣ The long noncoding RNA megacluster, lnc-MGC hosts nearly 40 miRNAs within a microRNA cluster called the miR-379 cluster. Smad site and CHOP site are binding sites for SMAD and CHOP transcription factors in the promoter region of lnc-MGC. b ∣ Expression of lnc-MGC, is induced by hyperglycaemia through the actions of TGF-β, which induces the binding of transcription factors such as SMADs. The resultant induction of miR-379 and miR-494 (which are located in the miR-379 cluster) targets and reduces the expression of negative regulators of endoplasmic reticulum (ER) stress, such as EDEM3 and ATF3, which in turn induces activation of the transcription factor CHOP, which in turn increases the expression of lnc-MGC and the miR-379 cluster. This signalling circuit might facilitate the persistent expression of lnc-MGC and the miR-379 cluster even after blood glucose levels have been controlled by antidiabetic agents, providing a potential mechanism for metabolic memory. c ∣ Erasure of this mechanism of metabolic memory might be achieved with use of a locked nucleic acid (LNA)-modified chimeric antisense oligonucleotide (a GapmeR) that targets lnc-MGC. Such an approach would result in cleavage of the lnc-MGC RNA by RNaseH and stop the persistent expression of lnc-MGC and the miR-379 cluster induced by the signalling circuit. TSS, Transcription start site.

Fig. 5.. Approaches to generate locus-specific genetic changes as a therapy for diabetic kidney disease.

CRISPR-Cas9 is a tool that enables targeted editing of the genome. Briefly, the Cas9 endonuclease is delivered into cells with a guide RNA (gRNA) that is designed to bind Cas9 and the target gene of interest. Following targeted Cas9 cleavage, the DNA is repaired by the cellular repair machinery (through non-homologous end joining or homology-directed repair). The system can be modified to achieve various outcomes, including (a) locus-specific induction of point mutations or short deletions, (b) the control of expression of specific genes (by fusing Cas9 with transcriptional activators or transcriptional repressors), (c) specific substitutions (for example by fusing Cas9 to adenine deaminase, which converts adenine (A) to inosine (I), which pairs with cytosine (C) and will be eventually converted to guanine (G) during replication), and (d) replacement of a long fragment of the genome by homology-directed repair. DNA damage (double-strand breaks) induced by CRISPR-Cas9 system can be repaired by homology-directed repair if the homologous template is close to the site. In this process, if an extra sequence is inserted between left arm and right arm of the homologous template (including extra sequences), the targeted sites can be replaced with the desired extra sequence.

Fig. 6.. Locus-specific epigenetic changes as potential therapy for diabetic kidney disease.

In addition to introducing locus-specific genetic changes, the CRISPR-Cas9 system can also be used to introduce locus-specific epigenetic changes, including (a) the induction of DNA methylation (by fusing Cas9 with DNA methyltransferase) or demethylation at specific sites (by fusing Cas9 with ten-eleven translocation (TET) proteins that convert 5-methylcytosine into 5-hydroxymethylcytosine) and (b) the induction of histone modifications, including histone acetylation (by fusing Cas9 with acetyltransferase), histone deacetylation (by fusing Cas9 with histone deacetylase), histone methylation (by fusing Cas9 with histone methyltransferase), and histone demethylation (by fusing Cas9 with histone demethylase). Such changes of epigenetic marks might facilitate the erasure of epigenetic memory and improve long-term outcomes for patients with diabetic kidney disease. gRNA, guide RNA.

Fig. 7.. Epigenetic and RNA profiling to support precision medicine.

Epigenetic changes such as DNA methylation and histone modifications in patients represent potential biomarkers for particular renal diseases, including diabetic kidney disease (DKD). Locus-specific correction of such epigenetic changes using the CRISPR-Cas9 system could be a future avenue for patient-specific treatment. Circulating RNAs (including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs)) in biological fluids, such as blood and urine, or biopsy tissue are also potential diagnostic biomarkers for DKD. Synthetic chemical inhibitors of specific RNAs (for example, antisense oligonucleotides (for example, GapmeRs) could also facilitate patient-specific treatment.