Epigenetics in kidney transplantation: current evidence, predictions and future research directions

Abstract

Epigenetic modifications are changes to the genome that occur without any alteration in DNA sequence. These changes include cytosine methylation of DNA at CpG dinucleotides, histone modifications, microRNA interactions, and chromatin remodeling complexes. Epigenetic modifications may exert their effect independently or complementary to genetic variants and have the potential to modify gene expression. These modifications are dynamic, potentially heritable, and can be induced by environmental stimuli or drugs. There is emerging evidence that epigenetics play an important role in health and disease. However, the impact of epigenetic modifications on the outcomes of kidney transplantation is currently poorly understood and deserves further exploration. Kidney transplantation is the best treatment option for end stage renal disease, but allograft loss remains a significant challenge that leads to increased morbidity and return to dialysis. Epigenetic modifications may influence the activation, proliferation, and differentiation of the immune cells, and therefore may have a critical role in the host immune response to the allograft and its outcome. The epigenome of the donor may also impact kidney graft survival, especially those epigenetic modifications associated with early transplant stressors (e.g., cold ischemia time) and donor aging. In the present review, we discuss evidence supporting the role of epigenetic modifications in ischemia reperfusion injury, host immune response to the graft, and graft response to injury as potential new tools for the diagnosis and prediction of graft function, and new therapeutic targets for improving outcomes of kidney transplantation.

Current Challenges in Kidney Transplantation

Successful kidney transplantation (KT) provides longer survival and better quality of life than dialysis for most end-stage renal disease (ESRD) patients (1, 2). Short-term kidney graft survival after KT has continuously improved in recent years (1). However, reports showed less impressive advances in long-term outcomes (3–9). Late graft loss continues to be a major problem after KT (3–11), mainly as consequence of death with a functioning graft and intrinsic allograft failure (or chronic renal allograft dysfunction (CRAD)). CRAD is a multifactorial clinical / pathological entity characterized by a progressive decrease in glomerular filtration rate (3).

Long-term function and outcome of the kidney allograft are determined by the overall cumulative injury, resulting from immunological and non-immunological (early and late) stressors (e.g., brain death, donor age, severity of ischemia-reperfusion (I/R) injury (12), nephron loss (13), infections, antigen and antibody responses causing ongoing renal tissue stress and inflammation), against a background of various donor and recipient risk factors that culminate in chronic interstitial fibrosis (IF) and tubular atrophy (TA) over time (3–11,15).

Molecular perturbations may precede not only graft dysfunction but also histological changes (16–29). Routine biopsies are used to assess organ quality and predict outcomes (12,30,31); however, multiple limitations are associated with this approach. Two recent studies (14,31) highlight the limitations of using donor organ biopsy results as the single criterion for declining a kidney offer. Similar limitations exist for the evaluation of late stressors that affect graft function leading to CRAD and graft loss over-time (32).

The lack of objective measures of organ quality, transplant function, and the inadequacies of clinical endpoints are major barriers to individualization of patient management (3). Recent advances in biotechnology have enabled the collection of large data sets to characterize the role of the genome, epigenome, transcriptome, proteome, and metabolome in KT (33–38). Multistage analysis that connects the molecular interactions and biological function of different kidney cells to renal physiology and pathology can be utilized to identify pathways of biological and clinical importance that are perturbed in disease processes (39–40). Integration of experimental data with donor and recipient clinical / demographic data through network based approaches will potentially elucidate the biological mechanisms associated with graft survival (41).

The epigenetic effects of environmental stressors, which can persist long after the removal of the injurious process, have yet to be adequately evaluated as diagnostic and predictor biomarkers of graft function (34). In this regard, due to the dynamic nature of the epigenome, its evaluation has high potential to identify targets for the development of new therapeutic strategies.

In the present review, we discuss the published evidence supporting the role of epigenetic modifications in I/R injury, host immune response to the graft, and graft response to injury, as potential tools for the diagnosis and prediction of graft function and new therapeutic targets for improving KT outcomes.

Association of Epigenetic Events with Disease

The epigenome represents the merging point between genetics and environment, where flexibility of the epigenetic code modifies the genetic code to determine the final phenotype (42). Functionally, epigenetics provides additional information about transcriptional control and plays a crucial role in physiological (42–44) and pathological conditions (45–47). Dynamic chromatin remodeling processes are required for the initial step in gene expression, which is regulated by epigenetic processes including DNA methylation (DNAm), histone modifications, and the action of small noncoding RNAs (Figure-1)(48,49). Epigenetic changes can modify gene expression by activating or silencing genes and determine which proteins are transcribed. In general, DNA is transcriptionally active when histones are acetylated and unmethylated, whereas deacetylated and methylated histones block gene expression (50–52). Methylation stops expression of genes through a number of mechanisms, including altering the arrangement and position of chromatin, blocking transcription factors from accessing the DNA, or recruiting repressors to the site of methylation (53,54).

Figure 1: Schema showing main components of epigenetic machinery and its role in regulation of gene expression.

(1) DNA methylation: DNA methylation primarily involves the covalent binding of a methyl group to the cytosine pyrimidine ring in cytosine-phosphate diester-guanine (CpG) islands. CpG islands are high-density clusters of CpG dinucleotide; these are associated with gene promoters and are conserved across species. Methylation of the cytosine residues at CpG sites is catalyzed by DNA methyltransferases, which provide a unique epigenetic signature that regulates chromatin organization and gene expression. Methylation of cytosines in CpG dinucleotides is associated with inactive, condensed states of the chromosome. (2) Histone modifications: The core histones (H2A, H2B, H3 and H4), together with the 147 base pairs of genomic DNA wrapped around them, comprise the nucleosomes, which are the basic units of chromatin. The interactions between DNA and histones determine the degree of chromatin condensation and, consequently, of gene expression. (3) Small noncoding RNAs: Mature functional microRNAs of approximately 22 nucleotides are generated from long primary microRNA (pri-microRNA) transcripts. First, the pri-microRNAs, are processed in the nucleus into stem-loop precursors (pre-microRNA) of approximately 70 nucleotides by the RNase III endonuclease Drosha and its partner Pasha. The pre-microRNAs are then actively transported into the cytoplasm by exportin 5 and Ran-GTP and further processed into small RNA duplexes of approximately 22 nucleotides by the Dicer RNase III enzyme and its partner Loqacious (Loqs). The functional strand of the microRNA duplex is then loaded into the RNA-induced silencing complex (RISC). Finally, the microRNA guides the RISC to the cognate messenger RNA (mRNA) target for translational repression or degradation of mRNA.

RNA reduces gene expression after transcription by repressing translation of messenger RNA (mRNA) or increasing RNA degradation. Transcriptomic profiling has revealed numerous noncoding RNA sequences transcribed from the human genome. The discovery of RNA interference (RNAi) mediated by siRNA and microRNA (miRNA) led to a new paradigm in gene regulation by demonstrating that short complementary RNAs can directly regulate target mRNAs (55). MiRNAs play a central role in cellular differentiation, development, proliferation and apoptosis, in several cell types. Notably, a complex epigenetic network exists whereby even miRNA expression is under epigenetic control (55–58). These RNA molecules target specific mRNAs (58–60). MiRNAs form an extensive layer of post-transcriptional regulators of gene expression. Among their preferred targets are transcription factors and epigenetic regulators, which in turn regulate the expression of individual miRNAs. In the kidney, miRNAs are critical in the maintenance of glomerular homeostasis and hence RNA interference may be important in the progression of renal disease (61–63). Because miRNAs are short and resistant against cleavage by RNAses, they can be evaluated in biological fluids (e.g., plasma, urine, saliva) and archival samples (paraffin-embedded tissue, FFPE). This characteristic makes miRNAs attractive as biomarker candidates. Importantly, miRNAs can be evaluated by the same techniques used for the identification of mRNA, ranging from multiplexed PCR to microarrays and next-generation sequencing (NGS). These techniques (and their advantages and disadvantages) are fully reviewed in recent detailed reports. (64–66).

More recently, a new class of regulatory RNAs known as long non-coding RNA (lncRNAs) has emerged as an additional layer of the circuitry controlling gene expression (67–69). Specifically, in the immune system, lncRNAs exhibit dynamic expression in cell type-, developmental stage-, and context-specific manners to coordinate several aspects of immune function (68). LncRNAs can offer benefits as biomarkers, especially when they can be detected in biological fluids. Furthermore, as opposed to mRNA, the lcnRNA itself is a functional molecule and its expression level may be a better indicator of disease (69). Moreover, the highly specific expression patterns of lcnRNAs suggest that their expression signatures could be used for accurate disease diagnosis and classification. Future and ongoing research will provide additional information about the feasibility of lcnRNA biomarker application (69).

While epigenetic changes are required for normal development and health, they can also induce pathological conditions (43). Disrupting any of the systems that contribute to epigenetic alterations can cause abnormal activation or silencing of genes (70). Such disturbances are associated with complex diseases including cancer (48), syndromes including chromosomal instabilities (71), mental retardation (72), chronic kidney disease (CKD) (73,74), and many others (75–77).

The field of cancer epigenetics has flourished from in vitro and in vivo discoveries, and from human clinical and epidemiologic studies (48,54,78–79). In contrast, there are very limited studies evaluating the role of epigenetic modifications in solid organ transplantation (34). Epigenetic modifications can occur during chronic illness, like those associated with the original disease leading to ESRD in KT recipients (39,73,74,81–83). These modifications may persist after transplantation and potentially influence allograft outcomes. Epigenetic mechanisms influence the activation, proliferation, and differentiation of the immune cells involved in the host immune response to the graft (84). Additionally, they have been also associated with the kidney tissue response to chronic injury (85,86). In this regard, recent studies showed epigenetic differences in the enhancer regions of fibrosis-related genes in human kidney samples (87). It follows that detailed interrogation of the epigenome in transplant recipients and donors has the potential to provide further insight into the epigenetic modifications associated with allograft survival.

Robust biomarkers are needed to reduce the level of immunosuppressive therapy and to improve long-term transplant outcomes (3). The use of epigenetic biomarkers, mainly based on DNAm, is well established for the diagnosis and prognosis of tumors and has begun to be understood in other pathologies such as autoimmune diseases. In a recent review, Mlcochova et al. (88) discussed the studies focusing on detection and characterization of urinary miRNAs as potential biomarkers in bladder, prostate, and kidney cancers. More recently, the same group summarized the studies focusing on detection and characterization of urinary miRNAs as potential biomarkers in urologic cancers, nephrology, and cardiology (89). Indeed, epigenetic biomarkers found in peripheral blood or urine samples might become new tools to detect early graft injury and to predict graft outcome.

Epigenetics and Kidney Transplantation

Despite only recent introduction in the field of KT, epigenetic modifications are increasingly recognized to play a key role in the multiple biological processes that contribute to kidney allograft outcomes (34,84,88). Each individual has unique modifications of the epigenome. Epigenetics is therefore a powerful determinant of how an individual responds to a disease challenge (70). Both the recipient and the organ donor genotypes undergo continuous and dynamic epigenetics modifications, even before transplantation (90–93).

Epigenetic modifications in ischemia reperfusion injury

I/R INJURY is a complex phenomenon that occurs in diverse clinical settings including KT and is characterized by tissue hypoxia. During the ischemic period, tissues are deprived of oxygen and nutrients required to maintain normal metabolism and energy homeostasis (95). I/R injury causes a series of pathological responses including early and late inflammation and fibrosis, which leads to cell and organ graft injury (95–99). In KT, these lesions associate with impaired short-term allograft function (delayed graft function (DGF)) and long-term graft survival by leading to CRAD (95–99).

One of the principal causes of I/R injury is the inherent oxidative damage that occurs on reperfusion by the generation of oxygen and hydroxyl free radicals (100,101). The overproduction of these reactive oxygen species is a common underlying mechanism damaging various cellular components, including proteins, lipids, and DNA.

(a). DNAm studies in kidney I/R injury

Pratt et al. (91) established that modifications of methylated cytosines may occur as result of prolonged ischemia and from additional reperfusion injury in transplantation. Specifically, in a rodent model of KT, the authors investigated the demethylation of a specific CpG within the IFNγ response element resident in the promoter region of complement component 3 (C3) gene in the rat kidney. After I/R injury a significant change in the ratio of methylated to unmethylated cytosines at this site was observed. Although this study indicated that hypoxia leads to epigenetic changes triggering ischemic injury, it was limited to the evaluation of methylation only within C3 gene, while aberrant methylation occurs across the genome of transplanted tissues. It is expected that the availability of high throughput reactions for testing DNAm patterns across the human genome (e.g., microarrays, NGS) (102,103) will enable larger studies that may provide critical additional information in the near future (Table-1).

Table 1-.

Main techniques/platforms available for evaluation of major epigenetics changes

Technique	Epigenetic change	Target	Advantage	Disadvantage
Bisulfite treatment* followed by PCR	DNA methylation	Locus-specific DNA methylation	Fast Cost-effective Appropriate when looking a small number of genes	Limited information to few CpG sites Low-scale screening
Pyrosequencing	DNA methylation	High-throughput quantitative method	Fast. Sensitive. Use always same reagents, but primers.	Variation in number of reads obtained per sample. Lack of resolution in homopolymer regions. False positives.
Microarrays (Illumina 450K Infinium Methylation BeadChip)	DNA methylation	Cover 99% of all RefSeq genes and approximately 450,000 CpGs	Low-cost alternative for profiling large number of samples. Relatively standardized analysis, accurate results.	Reduced resolution compared with NSG. Bias may exist as consequence of specific probes and allelic specific differences.
Whole-genome bisulfite sequencing		Unbiased assessment of the profile of DNA methylomes	High resolution Large amount of data	Complex data analysis, highly dependent in software analysis Expensive (but cost going down)
N-ChIp	Histone modification, uses native chromatin, which is unfixed and nuclease digested.		High antigen specificity for the analysis of histones and their isoforms. PCR amplification of the immunoprecipitated DNA is not needed because DNA recovery is higher. Specificity of the binding is more predictable because the antisera is raised against unfixed proteins.	High concentrations of nuclease may overdigest the chromatin, leading to subnucleosomal proteins, thus hindering the detection of protein-DNA interactions. Not all the nuclease-digested proteins are solubilized. Thus, a fraction of the chromatin is retained with the nuclear pellet and eliminated from the assay. Chances of chromatin rearrangement during processing are likely.
ChIP-followed by high-throughput sequencing (ChIP-seq)	Histone modification	Single nucleotide. Limited only by alignability of reads to the genome; increases with read length; many repetitive regions can be covered	Possible multiplexing. Large genomes Less amplification required; single molecule sequencing without amplification is available	Low required amount of ChIP DNA (10–50ng)
ChIP-Microarray (ChIP-chip)	Histone modification	Array-specific, generally 30–1100 bp Limited by sequences on the array; repetitive regions usually masked out	Can be used to determine unknown DNA sequences that interact with a known protein with no prior knowledge of its target genes. Can detect a large number of target genes at a time. Increases the chances of detecting target promoters.	Genomic microarray of the mammalian system is difficult due to the large size. High (few μg) required amount of ChIP DNA
qPCR	miRNAs	Targeted microRNAs	Established method, sensitive and specific. Can be used for absolute quantification	Cannot identify novel microRNAs Only medium-throughput with respect to the number of samples processed per day
microarrays	miRNAs	Most identified microRNAs (total number varies with platform)	Established method. Fairly low-cost and high-throughput with respect to the number of samples that can be processed per day	Lower specificity than qRT-PCR or RNA sequencing. Difficult to use for absolute quantification. Limited to known/identified miRNAs.
NGS	miRNAs	Discovery and undiscovered microRNAs	High accuracy in distinguishing miRNAs that are very similar in sequence. Can detect novel miRNAs	Substantial computational support needed for data analysis. Cannot be used for absolute quantification.

^*Bisulfite conversion, also known as bisulfite treatment, is used to deaminate unmethylated cytosine to produce uracil in DNA and is considered the “gold standard” for downstream applications to assess DNA methylation status. NGS: next generation sequencing; qRT-PCR: quantitative polymerase chain reaction

In a new study, the same group of investigators evaluated if the loss of transcriptional repression of the C3 gene provides a reasonable explanation for the emphasized immunologic injury following prolonged ischemia of the allograft (92). This study contributes to the understanding that a transient ischemic insult may influence long term chronic pathological changes in clinical transplantation. However, additional studies are needed for corroborating the effect/consequence of methylation patterns after I/R injury on C3 gene and gene expression.

Mehta et al. (104) investigated aberrant methylation of two gene promoters (DAPK and CALCA) in urinary DNA from deceased (DD) and living donor (LD) kidney transplant recipients at 48 hours post-transplantation and compared with normal healthy controls. Transplant recipients were significantly more likely to have aberrant hypermethylation of the CALCA gene promoter in urine than healthy controls, but there was increased CALCA hypermethylation in the urine of DD versus LD transplants, implying a role of the ischemia in the methylation patterns. Furthermore, there was a trend toward increased aberrant hypermethylation of urine CALCA in patients with biopsy-proven acute tubular necrosis versus acute rejection (AR) and slow or prompt graft function. These results represent an important finding, but were not statistically significant, perhaps due to the small sample size. In addition to the limitation associated with the small sample size and gene-specific restricted analysis (only two gene promoters), the utility of DNAm patterns in DNA from urinary cell pellets as markers of kidney I/R injury needs to be further validated.

(b). Histone Acetylation Studies in kidney I/R injury: Possible new therapeutic interventions

Increase in histone acetylation, the levels of which are determined by the balance between the activities of histone acetyltransferase (HAT) and histone deacetylase (HDAC), generally stimulates gene transcription by relaxing the chromatin structure (53). The study by Marumo et al. (105), in which I/R injury was induced in a mice model, demonstrated that transient ischemia induces epigenetic changes characterized by a decrease in histone acetylation probably as a result of decreased in situ HAT activity in the ischemic period and subsequent down-regulation of HDAC5 that contributes partially to histone re-acetylation and bone morphogenetic protein-7 (BMP7) induction in the recovery period. These observations showed a novel function of HDAC5 as a sensor that conveys the cellular energy conditions to chromatin and also suggested HDAC5 inhibition as a potential therapeutic strategy for enhancing BMP7 expression that can lead to the regeneration of the damaged kidney.

The anti-inflammatory and antifibrotic effects of HDAC inhibitors (HDCAi) have recently been reported (106,107), and suggest their potential as a novel therapeutic strategy for treatment of kidney dysfunction. Although their inflammatory effects have only just begun to be elucidated, some HDCAi are already showing therapeutic promise in animal models of inflammatory diseases including I/R injury. However, because non-specific HDAC inhibitors were used in those in vivo experiments, current knowledge on the action of individual HDACs in kidney injury is very poor, limiting the clinical application. Kim et al. (107) showed up-regulation of plasminogen activator inhibitor type-1 (PAI-1) by release of HDAC11 from a specific region of the PAI-1 promoter in kidney tissue samples from BALB/c mice. This event occurs in an androgen -dependent manner, suggesting a sex-selective mechanism in kidney I/R injury, and possibly a novel and specific epigenetic target. Acute kidney injury (AKI) is a risk factor for progression to chronic kidney disease (108). However, no therapies have been proven to reduce post-injury fibrosis in patients with AKI. Recently, Novitskaya et al. (109) showed that treatment with the phenylthiobutanoic acid (PTBA) analog methyl-4-(phenylthio) butanoate (M4PTB) initiated 4 days after injury (injected intraperitoneally at 100 mg/kg daily starting 4 days after AA injection) accelerates functional recovery, reduces long-term tubular atrophy and interstitial fibrosis, and enhances regenerative repair of injured renal tubular epithelial cells in a mouse model of aristolochic acid induced- AKI. These initial findings establish groundwork for further studies to evaluate a possible way of decreasing inflammation by means of HDAC inhibitors in I/R injury.

(c). Functional roles of miRNAs in kidney I/R injury

Among the epigenetic mechanisms, regulation of gene expression through miRNAs is the most dynamic (110). Epigenetic regulators are strongly enriched among the predicted targets of miRNAs, which may contribute to the recognized importance of miRNAs for pluripotency, organism development and somatic cell reprogramming (56). Several studies have suggested a functional role for miRNAs in I/R injury (111). In studies of renal I/R injury models by Godwin et al. (112) and Wei et al. (113), several miRNAs including miR-21, miR-7, and miR-192 were shown to be upregulated, while others such as miR-322 were down-regulated. Specifically, global miRNA expression profiling on samples prepared from the kidneys of C57BL/6 mice that underwent unilateral warm ischemia revealed nine miRNAs differentially expressed following I/R injury when compared with sham controls (112). These miRNAs were also differently expressed following I/R injury in immunodeficient RAG-2/common gamma-chain double-knockout mice and therefore define a lymphocyte-independent signature of renal I/R injury. Moreover, miR-21 expression was shown to be increased in proliferating tubular epithelial cells, whereas knockdown of miR-21 in these cells resulted in enhanced apoptosis. These findings therefore define a molecular pattern of renal injury. In a subsequent study using bioinformatics tools, the same group highlighted the usefulness of this distinct miRNA expression pattern in I/R injury versus sham controls as a distinguishing biomarker using principal component analysis (114).

The role of miRNAs in renoprotection through delayed ischemic preconditioning was also recently investigated (115). Specifically, a 15-min renal ischemic preconditioning significantly increased the expression of miR-21 by 4 h and substantially attenuated I/R injury induced 4 days later. An anti-miR-21 given at the time of ischemic preconditioning knocked down miR-21 and significantly exacerbated subsequent I/R injury in the mouse kidney. Knockdown of miR-21 resulted in significant upregulation of programmed cell death protein 4 (PDCD4), a proapoptotic target gene of miR-21, and substantially increased tubular cell apoptosis. Silencing of miR-21 without ischemic preconditioning did not ameliorate I/R injury in this study. Recently, a study showed in an I/R mice model that upregulated miR-21 had lower plasma levels of blood urea nitrogen (BUN) and creatinine, lower histopathological scores and a decrease in PDCD4 mRNA and active caspase-3, caspase-8 proteins expressions, supporting that miR-21 has anti-apoptotic properties by suppressing the expression of PDCD4 gene and active caspase 3 and 8 fragments in the condition of renal I/R injury (116). The diverse effects of miR-21 on I/R injury are likely due to temporal changes in expression of miR-21 target genes and pathway involvement through the different stages of I/R injury pathology (117). Another group identified miR-127 to be consistently deregulated during I/R in vitro and in vivo in a rat model. MiR-127 was found to be regulated by hypoxia-inducible factor 1α (HIF-1α) bioinformatically and to target kinesin family member 3B (KIF3B) (118). However, the regulation of miR-127 by HIF-1α could not be confirmed experimentally in this study. Also, Bijkerk et al. (119) recently showed that miR-126 protected mice from post-ischemic acute renal injury.

MiRNA-enriched microvesicles (MVs) secreted by endothelial progenitor cells were shown to ameliorate I/R injury in the murine kidney (120). The RNA content of microvesicles was enriched in miRNAs that modulate proliferation, angiogenesis, and apoptosis. After intravenous injection following ischemia-reperfusion, the microvesicles were localized within peritubular capillaries and tubular cells. This conferred functional and morphologic protection from AKI by enhanced tubular cell proliferation, reduced apoptosis, and leukocyte infiltration. The renoprotection effect was lost after elimination of miRNAs using different strategies (i.e., RNase, nonspecific miRNA depletion of microvesicles by Dicer knock-down in the progenitor cells). Lorenzen et al. (121) demonstrated that miR-24 antagonism prevents renal reperfusion injury in vivo. The treatment with an antisense oligonucleotide targeting miR-24 before the induction of I/R injury resulted in a significantly improved kidney function. The authors attributed this effect to a decreased apoptotic response after the ischemic insult in the kidney via the target genes HO-1 and H2A.X as consequence of silencing of miR-24 (121). More recently, the same investigator tested whether circulating lncRNAs in plasma of critically ill patients with AKI at start of renal replacement therapy were deregulated and might predict survival (122). Specifically, a global lncRNA expression analysis using RNA isolated from plasma of patients with AKI, healthy controls, and ischemic disease controls was performed. This analysis revealed numerous deregulated lncRNAs in plasma samples of patients with AKI. lncRNA-array-based alterations were confirmed in kidney biopsies of patients as well as in plasma by quantitative PCR (qPCR). Circulating concentrations of the novel intronic antisense lncRNA TrAnscript Predicting Survival in AKI (TapSAKI) were detectable in kidney biopsies and upregulated in plasma of patients with AKI, supporting TapSAKI as a predictor of mortality in this patient cohort. This study represents one of the first reports in nephrology showing utility of lncRNAs as biomarker of disease and prognosis (122).

It is anticipated that pharmacological tactics could be developed using locked nucleic acids to target detrimental ncRNAs involved in I/R injury, thereby reducing the initial damage in the transplanted organ and consequently improving transplantation outcome (123–125).

Epigenetics and Allo-Immune Response

The immune system of the recipient is the key player in the allo-response to the graft (126,127), and epigenetic regulation maintains the balance of immune response (93,128,129). The immune response shaped after KT is a continuous interchange between the innate and adaptive immune systems. As reported by the OPTN & SRTR Annual Data Report 2012, short-term kidney graft survival after KT has continuously improved in recent years. This is mainly consequence of the decrease in the incidence of AR as consequently of the use of potent immunosuppressive drugs (1). However, acute graft rejection still is responsible for up to 12% of graft loss and may be generally classified into antibody mediated rejection (ABMR) or acute cellular rejection (ACR) (130).

The development and activation state of immune cells is dependent on a tightly regulated and integrated gene-expression program controlled by well-established transcription factors and chromatin-modifying complexes (131).

Naturally occurring CD25⁺CD4⁺ regulatory T (Treg) cells, which constitutively express the transcription factor Foxp3, are essential for the maintenance of immune self-tolerance and homeostasis by suppressing excessive immune responses harmful to the host (132). The majority of Foxp3⁺ natural Treg (nTreg) cells are produced by the thymus as an antigen-primed and functionally mature T cell subpopulation specialized for immune suppression. The main task of Foxp3⁺ nTreg cells is to migrate to inflammation sites and suppress various effector lymphocytes, especially helper T (Th) cell subsets: Th1, Th2, Th17, and follicular Th (Tfh) cells (133,134). The balance between these immune responses directly affects the graft survival. The development of these responses is coordinated by a diverse group of functionally specialized cells and is highly influenced by the cellular microenvironment in which they are triggered (131–133). These cells lineages are established during hematopoietic differentiation through the interplay of multiple transcription factors and epigenetic modifications (133). Epigenetic patterns can be altered under certain environmental conditions, changing chromatin structure and modifying gene transcription, contributing to cell plasticity and, finally, determining the strength of post-transplant immune responses (131–135). As an example, a recent published review discusses the diverse set of mechanisms directing the functional instability or reinforcement of the Treg phenotype that include intrinsic epigenetic and transcriptional programs as well as miRNA and posttranslational regulation (134).

A common phenomenon across these different mechanisms has been the probable or experimental evidence of the control by environmental contributions. Further understanding of how the heterogeneous Foxp3⁺ Treg population responds to such influences may very well reconcile observations of staunch Treg lineage stability with profound Treg dysfunction in the face of certain inflammatory microenvironments (134). Thus, defining and modifying the “normal” epigenetic setting of cells of the immune system could have important clinical consequences for the diagnosis and treatment of kidney transplant recipients.

a). Role of DNAm patterns in immune-related genes and cells

The identification of DNAm patterns in immune -related genes could result in new genetic-based post-transplant biomarkers with applications in clinical practice. For example, Tregs cells play an important role in the maintenance of transplantation tolerance (133–135). Foxp3, a member of the forkhead/winged-helix family of transcription factors, acts as the master regulator for Treg development and function (132,136). Epigenetic markers have been reported at the Foxp3 locus and sub-populations of Treg cells differ in the methylation pattern of the Treg-specific demethylated region (TSDR) in the FOXP3 gene (137). In particular, CpG dinucleotides at the Foxp3 locus are methylated in naive CD4+CD25− T cells, activated CD4+ T cells, and TGF-beta-induced adaptive Tregs, whereas they are completely demethylated in natural Tregs. Recent studies suggest that Treg adoptive therapy may provide an effective approach to control alloreactivity (133,134). Moreover, demethylation of FOXP3 gene in biopsies from kidney transplant patients with subclinical rejection has been associated with a better long-term allograft outcome (137,138).

Wood et al. (139) recently reported that Treg-TSRD-demethylated CD4+ lymphocytes amplify the association of higher proportions of Tregs in KT patients with cutaneous squamous cell carcinoma (cSCC) compared to those without cancer. Interestingly, they found that the proportion of FOXP3+ cells was less than the proportion of TSDR-demethylated CD4+ cells. Specifically, in a cohort of long-term KT patients with and without skin cancer, the authors evaluated whether the peripheral blood immune phenotype remains stable over time and studied TSDR-demethylated cells. Despite the interesting findings of this study, the use of total PBMC instead of sorted Treg may represent a limitation of the study.

A challenge for the treatment of AR and induction of tolerance is to focus on cell therapy with Treg cells. A major inconvenience, however, is the loss of FoxP3 expression and reprogramming into effector T cells secreting IL-17 and IFN-ɣ in a pro-inflammatory microenvironment. Memory Tregs can become IL-17+ cells through DNA demethylation of the RORC locus. (140). However, although Treg cells can lose FoxP3 expression, they maintain the demethylated status of TSDR and retain the ability to reactivate FoxP3 expression and the suppressive function upon activation (141). Thus, the DNAm status of TSRD could be useful for specifically identifying suppressor Treg cells during clinical follow-up of transplanted patients (142).

Transplanted patients with a higher frequency of donor-specific memory T cells have a higher risk of alloreactive memory T cell-mediated rejection representing a barrier to tolerance (143). During memory T cell formation, epigenetic changes in transcription factors, cytokines and other molecules are essential for controlling the transcriptional profiles and function of these cells (144). For example, in memory CD4+ T cells, demethylation of the CCR6 gene allows the stable expression of this chemokine, enabling migration of these cells toward the renal proximal tubular epithelial cells, where they accomplish their function (145). More recently, Mazzoni et al. reported at the epigenetic level the origin of non-classic Th1 cells from Th17 cells, identified in the RORC2 and IL17A methylation status a novel tool for their distinction from classic Th1 cells, and demonstrate that RORC2-expressing cells are only a minority in the subset of CD4(+)CD161(+) naive Th cells, which are known to contain all Th17 cell precursors (146).

Depending on the early activation of natural killer (NK) cells after solid organ transplantation and the type of NK cell receptors expressed, these cells can promote rejection or tolerance (147). Several studies have demonstrated that epigenetic modifications modulate the cytolytic activity of NK cells regulating the expression of some NK cell receptors (KIR, NCRs and NKG2D) and cytotoxic molecules (GRZ and PRF) (148,149).

There is clear evidence of the role played by B cells, plasma cells and their secreted antibodies in the ACR and in ABMR (150). Differentiation of hematopoietic stem cells into antibody-producing B cells requires a complex epigenetic regulatory mechanism that produces changes in chromatin structure throughout B cell development (151,152). Altered DNA demethylation patterns within gene bodies outside of CpG islands are essential for the differentiation and function of B cells (153). Early in development, demethylation of transcription factors and their target genes is essential to establish a functional expression pattern in B cells and to initiate immunoglobulin V(D)J recombination (154). In later stages, naive B cells migrate to germinal centers, where they are activated by antigen and become antigen-experienced plasma cells producing antibodies or memory B cells (155). The activation-induced by cytidine deaminase (AID) enzyme resulting in diversification and high-affinity antibody production, is involved in the active DNA demethylation process (156,157). Further studies of these mechanisms are needed to determine whether targeting these enzymes could help attenuate antibody production.

A recent study by Rodriguez et al. (158) examined global DNAm levels and promoter specific methylation in a cohort of 47 patients before and after allogenic hematopoietic cell transplant (HCT). The authors demonstrated that DNAm analysis in HCT provides useful information that, if confirmed, may be useful as a diagnostic tool of relevant clinical parameters. This preliminary evidence support further evaluation of DNAm patterns in immune-associated genes and its potential use as biomarkers.

(b). miRNAs in AR and tolerance induction

The potential of miRNAs as biomarkers for diagnosis of AR and response to therapy might have critical impact in the transplant field. Sui et al. (159) described the first comparison of miRNA expression profiles between AR and controls. The authors identified 20 miRNAs differently expressed in AR after renal transplantation. However, the study had some limitations, including small sample size and use of pooled samples. Recently, Anglicheau et al. (160) proposed that intragraft levels of miR-142–5p, −155, −223, −10b, −30a-3p, and let-7c were diagnostic for AR in human renal allografts.

Sui et al. (161) in a subsequent study investigated the mechanisms of rejection using data integration among protein, mRNA miRNA and long ncRNA in biopsies of three patients with AR. The authors identified five transcription factors that were activated in biopsies with AR, which correlated with 12 miRNAs and 32 long ncRNAs. This study approach reveals the need of data integration for evaluating the complexity associated with molecular regulation of allo-response to the graft, and an example for future larger studies when using data integration.

Lorenzen et al. (162) evaluated whether miRNAs are also detectable in urine and may serve as new predictors of outcome in KT patients with AR. The miR-10a, miR-10b and miR-210 were strongly deregulated in urine of patients with AR. Betts et al. (163) identified a miRNA signature that associates with AR in serum samples. More recently, Danger et al. (164) studied expression profiling of miRNAs in peripheral blood mononuclear cells (PBMC) of KT recipients with chronic ABMR or stable graft function. Among 257 expressed miRNAs identified as differentially expressed in PBMC, 10-top ranked miRNAs associated with chronic ABMR were selected and used for classifying patients using a principal component analysis. In an independent validation using PBMC from a new set of patients, miR-142–5p was validated as marker of ABMR. Additionally, miR-142–5p was increased in paired kidney biopsies of patients with chronic ABMR. These data suggest that miR-142–5p could be used as a biomarker in chronic ABMR either in PBMC or biopsies. Moreover, functional assessment of these findings may improve our understanding of chronic rejection mechanisms. Also, validation in larger cohorts of patients should establish its diagnostic power as a biomarker for chronic ABMR.

While, the described studies show differential expression patterns of miRNAs in AR after KT, mechanistic evaluations of the specific pathways that associate with the affected miRNA profiles are still needed. Also, most of these studies are limited by small sample size and lack of reproducibility of the identified miRNAs among studies. This last issue can be explained, in part, by the use of different platforms and miRNA panels in the different studies (Table-1).

Additionally, miRNAs are essential for the maintenance of immune homeostasis and self-tolerance. Thus, a better understanding of how miRNAs regulate the immune response could introduce new tools for manipulating graft immunity. Danger et al. (165) identified overexpression of miR-142–3p in PBMC from tolerant kidney transplant recipients compared with stable recipients. Moreover, the authors showed the effects of miR-142–3p on B-cell-specific gene expression. Many of the identified genes were reported as potential miR-142–3p targets and previously identified in the blood of operationally tolerant patients (166).

Graft response to injury: Epigenetic modification and kidney graft fibrosis

Generally, the development of late graft failure is considered a variant of CKD, sharing common events of decline of kidney function and renal fibrosis (167). Recent studies provide preliminary evidence that epigenetic variations might determine the individual susceptibility of patients to develop chronic progressive kidney disease (47,72,73,86). Each individual has unique modifications of the epigenome, making epigenetics a powerful determinant for evaluating inter-individual variability response to a disease challenge (72, 168). Fibrosis is considered the common pathway of chronic progressive kidney disease. Fibrogenesis is the result of complex interactions among the different involved cell types which is coordinated by an extensive network of growth factors and signaling pathways (169–171). There is emerging evidence for the role of epigenetics influence on gene expression and kidney fibrogenesis, therefore providing unique new opportunities to further explore the diagnostic, predictive and therapeutic potentials of these variations. The reversibility of epigenetic alterations and the ready availability of small molecule inhibitors of these enzymes provide a potential future therapeutic strategy that could antagonize the profibrotic phenotype of fibroblasts.

(a). Methylation patterns and kidney fibrosis development

Allograft fibrosis is a pathologic scarring process that characterizes CRAD development. Activated fibroblasts are considered main mediators of renal fibrosis (172). In preclinical studies, it has been described that methylation determines fibroblast activation and fibrogenesis in the kidney (84). Bechtel et al. (85) showed that hypermethylation of RASAL1 encoding an inhibitor of the Ras oncoprotein, associates with the perpetuation of fibroblast activation and fibrogenesis in the kidney.

Clinical studies also show the role of DNAm in kidney fibrosis progression. Stenvinkel et al. (173) evaluated peripheral blood cell DNAm in well-defined cohorts of renal patients to evaluate the association between renal function, hyperhomocysteinemia, inflammation and aberrant DNAm. A main finding of this study is the association between surrogate markers of inflammation and DNAm in peripheral blood leucocytes. While stable CKD patients without evidence of inflammation had methylation patterns comparable with age- and gender-matched controls, CKD stage 5 patients exhibited DNA hypermethylation.

Recently, in one of the largest studies evaluating methylation patterns in CKD , Smyth et al. (174) evaluated a total of 485,577 methylation sites in 255 individuals with CKD and 152 individuals without evidence of renal disease. The analysis focused on statistically significant differential methylation of candidate genes with known biological function in CKD. Strong biological candidates for CKD that showed statistically significant differential methylation include CUX1, ELMO1, FKBP5, INHBA-AS1, PTPRN2, and PRKAG2 genes. Of particular interest, PRKAG2 has been associated with CKD by genomic DNA SNP studies (145) as well as DNAm and gene expression data (175). A limitation of this study is that blood-derived DNA was used rather than DNA obtained from affected and unaffected kidneys. Additionally, aiming to evaluate the relationship between gene expression and methylation status for the significant identified genes, blood for only four individuals; two cases with CKD and two controls with no evidence of kidney disease, was used.

Sapienza et al. (80) examined potential epigenetic biomarkers for CKD progression by comparing site-specific DNAm levels from saliva samples in more than 14,000 genes between African American (AA) and Hispanic ESRD patients with Type 2 diabetes and diabetic patients without nephropathy. They identified 187 genes that were differentially methylated between the two groups on at least two CpG sites in each gene. Of the 187 genes, 39 genes have been reported to be involved in kidney development or diabetic nephropathy. However, the study group contains mainly AA patients, and specific methylation pattern effects associated with race and kidney function will need to be addressed in a larger study.

Recently, Wing et al. (176) studied the genome-wide DNAm pattern associated with rapid loss of kidney function in 40 Chronic Renal Insufficiency (CRIC) study participants with the highest and lowest rates of decline in estimated glomerular filtration rate. The study identified differential methylation in CpG islands of several genes that are associated with rapid loss of kidney function. The most functionally relevant epigenetic signatures in subjects with a rapid decline in kidney function included key genes NPHP4, IQSEC1 and TCF3, which are involved in pathways known to promote the epithelial to mesenchymal transition. The authors mainly concluded that epigenetic modifications may be important in determining the rate of loss of kidney function in patients with established CKD. Although the study subjects were rigorously selected from a large pool of patients with varying level of renal function, the number of patients studied is small. Importantly, there are epigenetic differences between tissues and the epigenetic changes that should be studied in specific renal cell types.

(b). HDACs and kidney fibrosis

The pathogenesis of many kidney diseases is characterized by dysregulation of cellular proliferation, leading to fibrosis. The pathogenic mechanisms of tubulointerstitial fibrosis are diverse and complex (171,172). However, key elements include activation of TGF-β signaling in fibroblasts and infiltration with inflammatory cells which promote tissue injury and fibrosis. Activation and proliferation of renal fibroblasts are stimulated by a variety of growth factors and cytokines, such as transforming growth factor, platelet-derived growth factor, fibroblast growth factor, and interleukin-6. Several intracellular signaling pathways, including the signal transducer and activator of transcription3 (STAT3) pathways, are activated in response to those growth factors/cytokines.

Since HDAC inhibition (HDACi) has been shown to be anti-fibrotic in the lung, liver, and skin (177,178), it is therefore not surprising that HDACis have been shown to have anti-fibrotic effects in models of chronic kidney disease (179). It has been demonstrated (179,180) that HDAC1 and HDAC2 play an important role in regulating proliferation of renal interstitial fibroblasts and expression of multiple cell cycle proteins. Further, HDAC1 and HDAC2 contribute to modulation of STAT3 tyrosine phosphorylation/activation and STAT3 mediates the proliferative effect of HDACs. Inhibition of HDAC1/2 promotes STAT3 acetylation. Acetylated STAT3 leads to its dephosphorylation/inactivation and subsequently antagonizes transcriptional regulation of the target genes associated with cell cycle regulation and proliferation. These findings suggested that targeting HDAC1/2 may be a promising strategy to attenuate progression of kidney diseases associated with renal fibrosis.

Additionally, it has been reported that treatment with histone deacetylase inhibitor Trichostatin A (TSA) prevents TGF-β1-dependent responses in cultured human renal tubule epithelial cells (RTECs) (181). Cells treated with TSA still demonstrated Smad protein phosphorylation, which relays TGF-β1 signaling to the nucleus, indicating that TSA blocks TGF-β1’s effects downstream of these factors.

In a separate study, Marumo et al. (182) also observed an increase in the expression of HDAC1 and HDAC2 and a decrease in histone acetylation in kidneys injured by ureteral obstruction. Treatment with a TSA attenuated macrophage infiltration and fibrotic changes. These results suggest that increased expression of HDAC1 and HDAC2 contribute to the production of CSF-1, macrophage infiltration, and other profibrotic responses in response to injury and implicate a potential of HDAC inhibition in reducing inflammation and fibrosis in tubulointerstitial injury.

Recently, a study established that Valproic acid (VPA) treatment ameliorates the renal injury and fibrosis in diabetic kidney by preventing the myofibroblast activation and fibrogenesis via HDAC inhibition and associated mechanisms, thereby improving the profibrotic and anti-fibrotic protein balance (183)

Currently, the profile of HDAC-modulated proteins in the setting of fibrosis is not clear. This knowledge is not only important for further understanding of the mechanism by which HDACs induce tissue fibrosis but also helpful to develop HDAC inhibition as a novel therapeutic strategy against fibrosis development in diseases such as CRAD.

(a). miRNAs and allograft fibrosis development

After acute kidney injury (e.g.; IRI, inflammation), damaged tubular epithelial cells initiate a wound-healing program. A key determinant of successful kidney repair is the ability of tubular epithelial cells to proliferate and replace damaged cells after injury (184,185). Transforming growth factor beta (TGF-β) and its downstream signaling cascades is a well-established mechanism that is linked to renal fibrosis in animal models, but its dysregulation has also been shown in native and transplant kidneys (186–191). Critically, inhibition of TGF-β ameliorates renal fibrosis independently of etiology (192).

In cases where the kidney injury is persistent (e.g., uncontrolled alloresponse, CNIT), the production of TGF-β1 is significantly stimulated (Figure-2). If the effect of the stressor continues over time, TGF-β1 production persists and contributes to downstream epigenetic modifications in fibroblasts that slowly transform these cells into activated myofibroblasts (83–85). Myofibroblasts proliferate and progressively secrete collagen in a growth factor–independent manner (83–85).

Figure 2: Effect of miRNAs in the regulation of mechanisms related to graft injury and reparation.

Epigenetic modifications, including altered patterns of miRNAs, affect the donor and recipient early in the kidney transplant process. I/R injury has been associated with differential expression of miRNAs. miR-21 is among the most highly up-regulated miRNA in acute kidney injury (106–108). After KT, acute process such as ACR, ABMR, and CNIT also associate with specific miRNA profiles that regulate the expression of hundreds of genes from the immune system. These miRNAs affect both innate and adaptive immune response to graft (132–140). The final outcome of the graft depends on the ability to repair and remodel. Persistent injury to the allograft leads to chronic inflammation and fibrogenesis, resulting in CRAD (with IF/TA) and loss of graft function. MiRNAs have been associated with fibrosis (166–169). MiRNAs can influence tissue fibrogenesis through various mechanisms. miRNAs can be essential downstream components of both fibrogenic and fibrosis-suppressive signaling pathways, and changes in miRNA expression directly affect the biological response following activation of these pathways. Three TGF-β-regulated miRNA families, miR-21, miR-200, and miR-29 have been shown to modulate renal fibrosis. MiR-21, through a feed-forward loop, amplifies TGF-β signaling and promotes fibrosis. Conversely, miR-200 and miR-29 reduce fibrosis by inhibiting epithelial-to-mesenchymal transition and preventing the deposition of extracellular matrix, respectively (170–175). Inhibition of miR-21 expression or augmenting miR-29 expression prevents kidney fibrosis in mice (170, 173).

Recent studies reveal that TGF-β also regulates several miRNAs that are involved in kidney fibrosis (193–196). Moreover, there is increasing evidence that some specific miRNAs can coordinately regulate several fibrosis-associated networks (197,198).

MiRNAs can influence tissue fibrogenesis through various mechanisms (197,198). MiRNAs can be essential downstream components of both fibrogenic and fibrosis-suppressive signaling pathways, and changes in miRNA expression directly affect the biological response following activation of these pathways (198). For example, the miR-29 family members regulate extracellular matrix (ECM) production (199). These miRNAs are consistently down-regulated when fibrosis occurs and their low expression correlates with up-regulation of ECM-related genes (200).

Members of the miR-200 family are down-regulated after TGFβ–induced epithelial-mesenchymal transition (EMT) and act as protectors of the normal epithelial phenotype (201). During EMT, polarized epithelial cells lose cell-cell sand cell-basement membrane interactions, and acquire properties such as increased migratory capacity. TGF-β signaling, through activation of mesenchymal transcription factors such Zeb1 and Zeb2, is a potent inducer of EMT. Contrarily, miR-200s are also predicted to repress TGF-β2, which may inhibit EMT and prevent fibrosis (202,203). However, as consequence of controversy about the existence of EMT in vivo, additional in vivo studies are needed to clarify the role of miR-200 and EMT in the kidney.

Oba et al. (204) showed that the miR-200 family is up-regulated after ureteral obstruction in mice, with miR-200b being strongly induced. Also, the injection of miR-200b precursor was able to ameliorate tubulointerstitial fibrosis in the obstructed kidneys. We recently sought to establish a miRNA signature in urinary cell pellets from KT patients diagnosed with CRAD (205). Twenty-two miRNAs were found to be differentially expressed in urine samples from patients graft biopsies with IF/TA, with miR-200 being down-regulated. We then selected twelve miRNAs and longitudinally evaluated them in urine samples of an independent set of patients, at two time points after KT. This independent set of patients included KT recipients classified as progressors versus non-progressors to CRAD based on histological findings and graft function at 24 months post-KT. A subset of these miRNAs, including miR-200b and miR-200*, was found to be differentially expressed in samples from patients progressing to IF/TA early after KT before histological allograft injury was evident.

As with miR-200, TGF-β signaling has also been shown to regulate miR-21. TGF-β promotes miR-21 synthesis by increasing transcription and enhancing post-transcriptional processing of the miRNA (206). In turn, miR-21 inhibits Smad7, an inhibitory Smad, leading to amplification of TGF-β signaling resulting in a fibrotic response (207). Lin et al. showed that SMAD7 is a direct target of miR-21, and overexpression of miR-21 may inhibit the proliferation of rat renal tubular epithelial cells (209). MiR-21 also promotes fibrosis through activation of ERK-signaling pathway, resulting in inhibition of apoptosis and promoting proliferation of fibroblasts (206,209).

Ben-Dov et al. (210) identified 16 miRNAs in samples from KT patient with IF/TA, with miR-21 being up-regulated. Recently, we identified 56 miRNAs, including miR-21, that were differentially expressed in patients with CRAD and IF/TA (211). These initial studies show that miR-21 also has a role in fibrosis development in KT patients.

Future directions

Epigenetics is an expanding field in transplantation. Epigenetic modifications play a key role in biological processes that contribute to allograft function and outcomes. Transplantation is a complex model because two genomes (the donor organ and the recipient) with independent epigenetic changes are leading to a common phenotype. There are several ways where these effects appear to be manifested. The immune system of the recipient plays a major role in the allo-response to the graft. The increasing knowledge of the epigenetic mechanisms involved in regulating the immune response will result in a more comprehensive understanding of the immunological processes involved in rejection and allograft tolerance. Consequently, in the coming years the identification of epigenetic alterations that occur during immune system deregulation after transplantation will lead to the development of new biomarkers and targets for novel interventions. Additionally, the role of epigenetics in kidney fibrosis development has been reported (44,73,86). The response of the graft to the injury is mainly under control of the donor genotype and its epigenome. The balance between these interactions will conduct to the overall graft and patient outcome. A better understanding of these epigenetic modifications in both, donors and recipients, may lead to identification of biomarkers of graft quality, improvement in transplant recipient selection, and progress in overall outcome prediction. Additionally, new therapeutic interventions can be expected (after refining drug specificity), as a result of the expanding research in epigenetics in kidney transplantation.

Both the donor and recipient genomes experiencing different epigenetic modifications influence graft function and its final outcome. Further evaluation of epigenetic modifications in both donors and recipients may lead to identification of biomarkers of graft quality, improvement in transplant recipient selection, and advancement in overall outcome prediction. The advances in high throughput technology and improved bioinformatics tools will allow integrative data evaluation of the components of epigenetic machinery and its influence on the human kidney transplant organ.

Further evaluation of epigenetic modifications have the potential to lead to tissue specific biomarkers (kidney miRNA profiles, CpG patterns) for diagnosis, risk stratification, and prediction of pathological conditions that may affect the graft. Moreover, these modifications can result in non-invasive (miRNA, DNA free), combined with rapid, specific and quantitative methods. However, limitations associated with biomarkers will associate with multicellular composition of peripheral blood (requiring previous cell separation for specific analyses (i.e., pure T cells), laser micro-dissection for tissue samples), identification of resident cells versus circulating cells in the graft, among others.

Furthermore, novel and specific or targeted therapeutic interventions can be developed as a result of the expanding research in epigenetics in KT. Additional studies will determine whether epigenetic treatments may one day become a valid therapeutic strategy for modulating the balance between immunity and tolerance. It will be also critical for identifying targets (lncRNAs and miRNAs) that are involved in I/R injury and regulatory mechanisms leading to graft fibrosis as new therapeutic targets.

Figure 3: Epigenetics modifications in kidney transplantation.

The kidney transplant model is characterized by the combination of two genotypes (donor and recipient). Each genome is affected for epigenetic modifications associated with pre-transplant conditions (e.g., chronic illness, aging) and also peri-surgical factors (e.g., ischemia, ischemia reperfusion injury). Post-transplantation, the recipient immune system (also characterized by a unique epigenome) is responsible for the magnitude of the allo-response. On the other hand, the donor graft is responsible of the intensity of the response to the chronic injury with consequent inflammation, tissue reparation, and fibrogenesis that will conduct to nephron loss. The balance between these interactions will conduct to the overall graft and patient outcome. A better understanding of these epigenetic modifications in both, donors and recipients, may lead to identification of biomarkers of graft quality, improvement in transplant recipient selection, and progress in overall outcome prediction. Additionally, new therapeutic interventions can be expected (after refining drug specificity), as a result of the expanding research in epigenetics in kidney transplantation.

Abbreviations

ABMR

antibody mediated rejection

ACR

acute cellular rejection

AKI

acute kidney injury

AR

acute rejection

CIT

cold ischemia time

CKD

chronic kidney disease

CNI

calcineurin inhibitor

CRAD

chronic renal allograft dysfunction

DGF

delayed graft function

DNAm

DNA methylation

FFPE

paraffin-embedded tissue

HAT

histone acetyltransferase

HDAC

histone deacetylase

IF

interstitial fibrosis

I/R

ischemia reperfusion

KT

kidney transplantation

lncRNA

long non-coding RNA

miRNA

microRNA

NGS

next-generation sequencing

PBMC

peripheral blood mononuclear cells

TA

tubular atrophy

Treg

Lymphocyte T regulator