Acute kidney injury (AKI) and chronic kidney disease (CKD) are the most severe consequences of kidney injury. AKI is commonly defined by a sudden (within 48 h) increase in serum creatinine and is usually transient (Kidney Disease, 2012). However, it may be as severe as to require renal replacement therapy and is associated with an increased risk of death. CKD is defined by abnormalities of kidney structure or function that persist for longer than three months and have consequences for health (Kidney Disease, 2013; Perez-Gomez et al., 2019). CKD may progress to end-stage kidney disease requiring renal replacement therapy and is associated with an increased risk of premature death. Indeed, CKD was among the fastest growing causes of death worldwide and it is estimated that it will become the second most common cause of death within the next century in some countries (Ortiz et al., 2019). AKI and CKD are considered interconnected syndromes, as CKD predisposes to AKI and AKI may accelerate CKD progression (Chawla et al., 2014). AKI was thought to be followed by complete recovery of kidney structure or function. However, the repair process may be unsuccessful leading to engagement of pathways promoting transition to CKD. In this regard AKI and CKD share a number of pathogenic processes, including inflammation and parenchymal cell death. Additionally, kidney injury during AKI may initiate or aggravate the processes of capillary rarefaction and fibrosis that characterize CKD (Kida et al., 2013; Polichnowski, 2018; Yang et al., 2018). The clinical care of patients with AKI or CKD is marred by a paucity of effective therapeutic approaches. Thus, there is no therapy that prevents or accelerates recovery from AKI. Additionally, only a handful of drugs have been approved to slow the progression of CKD, including renin-angiotensin system blockers for proteinuric CKD and tolvaptan for polycystic kidney disease (Perez-Gomez et al., 2015). However, they slow but do not completely prevent CKD progression and do not promote regression. Thus, novel therapeutic approaches should be developed based on an improved understanding of the cellular and molecular pathogenesis of AKI and CKD and of the drivers of the AKI-to-CKD transition.

Epigenetic regulation refers to mechanisms that control gene expression without altering the nucleotide sequence. In general, epigenetic modifications are heritable during cell division although they are reversible and could be altered by age, disease, and environment. Epigenetic regulators include non-coding RNAs, and covalent modifications of histones and DNA. Histone modifications involve proteins responsible for the addition (writer), recognition (reader), and removal (eraser) of modifiers such as acyl-lysine modifications.

The main modification on DNA is methylation catalyzed by DNA methyltransferases (DNMTs). These are enzymes that methylate the 5-cytosine of CpG dinucleotides, frequently located in the first exons or near promoters (Mohtat and Susztak, 2010). DNA promoter methylation is a silencing mechanism that inhibits transcription factor binding through chromatin packaging or repressor recruitment (Beckerman et al., 2014).

Histones are positively charged and they associate with negatively charged DNA to package in condensed chromatin and form nucleosomes, which are the basic units of chromatin. Nucleosomes pack 147 DNA base pairs around a histone octamer composed of two H3-H4 histone dimers stabilized as a tetramer which is flanked by two separate H2A-H2B dimers (Luger et al., 1997; Davey et al., 2002). Histone 1 (H1) acts as a linker and completes the chromatosome by protecting internucleosomal linker DNA (Campos and Reinberg, 2009). There are more than 100 types of histone modifications which regulate gene expression, such as methylation, acetylation, phosphorylation, crotonylation, sumoylation, and ubiquitination (Susztak, 2014). However, the biological function of most of these modifications remains unknown. The best characterized consist of addition of acyl groups to specific amino acids, resulting in methylation, acetylation and crotonylation of lysine residues on histone H3.

Histone methylation requires the transfer of methyl groups to histone lysine or arginine residues by histone methyltransferases. Lysine residues can accept up to three methyl groups, resulting in mono-, di-, or trimethylated lysine, and arginine can accept up to two methyl groups, resulting in mono- or dimethylated arginine. Methyl groups can be removed by histone demethylases (Morera et al., 2016). The addition of methyl groups does not change histone charge, rather provides platforms for binding of transcription factors that may stimulate or suppress gene expression (Nguyen and Zhang, 2011).

Histone acetylation, the transfer of an acetyl group to lysine residues, changes the charge of histones and favors chromatin relaxation, facilitating binding of transcription factors to promoters and gene expression (Ramakrishnan and Pili, 2013). Histone acetylation is catalyzed by histone acetyltranferases (HAT) and it is reversible when histone deacetylases (HDAC) remove acetyl groups.

Histone lysine crotonylation (Kcr) was more recently described and consists in the transfer of crotonyl groups to lysine residues of histones, also conferring them negative charge (Tan et al., 2011). Recently, a role of histone Kcr was described in AKI (Ruiz-Andres et al., 2016a). We now review the functional relevance of histone crotonylation in tissue physiopathology with emphasis in kidney disease and the implications for the development of novel therapeutic approaches.

Both AKI and CKD are associated with cell death, inflammation, and diverse degrees of fibrosis. All of these could be regulated by epigenetic modifications. DNA methylation and histone acetylation and methylation are the most studied epigenetic mechanisms in renal injury, and evidence supports their functional relevance for kidney disease ( Table 1 ).

Global kidney DNA methylation was reduced in experimental AKI induced by ischemia-reperfusion injury (IRI) or cisplatin, and this was associated to the expression of inflammatory genes (Guo et al., 2017; Zhao et al., 2017). In this regard, DNA methylation contributes to silencing the expression of the nephroprotective genes Klotho (which has anti-inflammatory and anti-fibrotic properties) and RASAL1 (which has anti-fibrotic properties) in AKI. DNMTs targeting by genetic ablation or pharmacological inhibition decreased fibrosis and prevented the AKI-to-CKD transition in part because of preserved expression of Klotho and RASAL1 (Bechtel et al., 2010; Sun et al., 2012; Tampe et al., 2017).

Histone methylation, depending on context, lysine residue or extent of methylation may activate or repress gene transcription. Overall kidney histone methylation was increased in preclinical CKD induced by unilateral ureteral obstruction (UUO) or diabetic nephropathy [reviewed in (Fontecha-Barriuso et al., 2018)]. In this regard, the expression of the histone methyl-transferase, EZH2, is increased in UUO and in CKD patients and its pharmacological inhibition reduced preclinical fibrosis (Zhou et al., 2016). Similar results were found for the histone methyl-transferase, SMYD2 in clinical and preclinical PKD (Li et al., 2017). In AKI induced by IRI or endotoxin, increased histone methylation has been linked to the increased expression of pro-inflammatory and pro-fibrotic genes (Naito et al., 2008; Zager and Johnson, 2009). Moreover, histone methylation in the MCP1 and NGAL genes was detected in urine from AKI patients (Munshi et al., 2011).

Histone acetylation generally favors gene expression and it has been extensively studied in renal injury. Global changes in kidney histone acetylation were observed in experimental AKI (e.g. IRI, endotoxemia) and CKD (UUO, diabetic nephropathy). Histone acetylation increased during renal injury in most animal models analyzed (endotoxemia, UUO, diabetic nephropathy in db/db mice) (Sayyed et al., 2010; Huang et al., 2015; Hewitson et al., 2017). During ischemia reperfusion injury (IRI), histone acetylation presents a more complex regulation, it is transiently decreased during ischemia, but it recovers, or even increases, after reperfusion, and may persist increased for 3 weeks (Marumo et al., 2008; Zager et al., 2011). The impact of histone acetylation over the expression of specific relevant genes in AKI has been also studied, including the nephroprotective genes PGC1α and Klotho, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) and the inflammatory cytokine Interleukine-6 (IL-6), among others (Naito et al., 2009; Li et al., 2010; Ruiz-Andres et al., 2016b). The functional contribution of histone acetylation to kidney injury is supported by numerous preclinical studies in AKI (e.g. cisplatin, IRI) and CKD [e.g. UUO, polycystic kidney disease (PKD), diabetic nephropathy] models, and HDAC inhibitors were frequently nephroprotective (Fontecha-Barriuso et al., 2018). Related with histone acetylation, another emergent line of research is the study of the bromodomain and extraterminal (BET) protein family. BET proteins are readers that bind to acetylated lysines of histones and facilitate binding of transcription factors. BET targeting has been beneficial in preclinical kidney disease and ongoing clinical trials of the BET inhibitor apabetalone explore kidney function as a secondary outcome (Suarez-Alvarez et al., 2017; Fontecha-Barriuso et al., 2018).

These data show the complex regulation of epigenetic modifications in kidney diseases. The overall pattern of the different DNA and histone marks suggests that epigenetic modifications could favor the differential gene expression observed in kidney disease, opening the door for therapeutic strategies targeting epigenetic modulation.

The application of high-sensitivity mass spectrometry techniques recently identified lysine crotonylation (Kcr) as a novel histone lysine acylation (Tan et al., 2011). Histone crotonylation represents an evolutionarily conserved histone mark which consists in the addition of a crotonyl group from crotonyl-coenzyme A to lysine residues, that similar to acetylation, modifies the charge of the histones (Tan et al., 2011; Fontecha-Barriuso et al., 2018). Nonetheless, the genomic pattern and function of histone crotonylation differs from histone acetylation (Tan et al., 2011). Histone crotonylation mainly marks active promoters and potential enhancers, but the impact on gene expression is unclear, as it can activate or repress gene transcription (Tan et al., 2011; Fellows et al., 2018).

Histone crotonylation was initially well characterized in human somatic and male germ cells (Tan et al., 2011). Recently, histone crotonylation has been observed in different cells and tissues but its function over gene expression has been only explored outside the kidney in gametogenesis, reprogramming of pluripotent stem-cells and colon epithelial cells.

Kcr has been identified in non-histone proteins. Studies using specific antibody enrichment followed by high resolution mass spectrometry and intensive bioinformatics analysis have demonstrated that Kcr occurs in a large number of proteins, predominantly in nuclei, but also in the cytoplasm (Wei et al., 2017b; Wu et al., 2017; Xu et al., 2017). Crotonylation was promoted by exposure to crotonate or CBP/p300 overexpression (Wei et al., 2017b). Crotonylated non-histone proteins have been characterized in different cell types such as Hela cells, H1299 cells (Wei et al., 2017b; Xu et al., 2017), HTC 116 (Huang et al., 2018), and in tissues such as lung, kidney, liver, colon, uterus, ovary, and brain of mice (Tweedie-Cullen et al., 2012; Xu et al., 2017).

Crotonylation of non-histone proteins is thought to be important in various biological processes, such as RNA splicing, gene expression, chromatin organization, nucleic acid metabolism, and the cell cycle (Wei et al., 2017b). However, the requirement of crotonylation for most of these putative cellular functions has not been formally tested. HDAC1 is among the crotonylated non-histone proteins in HELA cells treated with crotonate and HDAC1 crotonylation leads to a reduced deacetylase activity (Wei et al., 2017b).

Kidney injury is probably the best characterized disease condition so far in terms of extent and function of histone crotonylation (Justo et al., 2006) ( Figure 2 ). Histone Kcr was observed in tubular cells from healthy mouse and human kidney and nephrotoxic AKI was associated with a global increase in kidney histone crotonylation (Ruiz-Andres et al., 2016a). Likewise, in cultured proximal tubule epithelial cell cultures, stimulation with the proinflammatory cytokine TWEAK, a driver of kidney injury (Sanz et al., 2008; Ruiz-Andres et al., 2015), increased global histone Kcr (Ruiz-Andres et al., 2016a). Additionally, crotonate supplementation increased global histone Kcr in cultured tubular cells and in the kidneys in vivo. Thus, levels of histone crotonylation in kidney tubular cells may be modified by cell stress or by crotonate administration.

The increased histone crotonylation in cells stimulated with TWEAK and in AKI kidneys was associated with decreased PGC1α and Sirt-3 expression and with increased CCL2 expression, a chemokine which contributes to kidney inflammation (Ruiz-Andres et al., 2016a). PGC1α and Sirt-3 have nephroprotective actions (Kong et al., 2010; Morigi et al., 2015; Fontecha-Barriuso et al., 2019). Besides, Sirt3 and PGC1α regulate each other. PGC1α is a transcription factor and a major regulator of gluconeogenesis, as well as driver of mitochondrial biogenesis, and Sirt3 is a mediator of the effect of PGC1α on mitochondrial biogenesis (Van Beneden et al., 2011). The increased histone crotonylation induced by crotonate administration in mice and in cultured tubular kidney cells resulted in an increased PGC1α and sirtuin-3 expression, as well as a decrease of CCL2 expression. That is, the systemic administration of crotonate prevents the decrease of PGC1α and sirtuin-3 expression, and the increase of CCL2 expression in mice with AKI and this was associated to protection from experimental nephrotoxic AKI and preservation of renal function (Justo et al., 2006). Taken together, the cell culture evidence that crotonate increased histone crotonylation and prevented proinflammatory responses while preserving nephroprotective gene expression, the fact that it had similar actions in vivo in AKI and that it protected from AKI, suggests that crotonate may have a potential therapeutic effect on kidney damage, and more specifically in AKI, by increasing histone crotonylation. If this hypothesis is correct, then the spontaneous global increase in histone crotonylation during AKI may be considered part of an adaptive mechanism, even allowing for potential discrepant level of crotonylation in specific genes. In this regard, the previously described beneficial effects of HDAC inhibitors such as valproic acid or trichostatin, in kidney damage (Moreno et al., 2011; Van Beneden et al., 2013), should be viewed in the context of their potential to induce histone crotonylation (Madsen and Olsen, 2012; Wei et al., 2017a; Kelly et al., 2018) and not just through the light of their impact on histone lysine acetylation. Some examples of beneficial effects of HDAC inhibitors that have been ascribed to modulation of histone acetylation but may in fact be related to increase histone crotonylation include preservation of the expression of the antiaging and nephroprotective gene Klotho despite the presence of Klotho suppressors such as inflammatory cytokines and albuminuria, and similar results were observed with PGC1α expression (Chen et al., 2018; Fernandez-Fernandez et al., 2018; Fontecha-Barriuso et al., 2019). This hypothesis should be addressed through specifically designed experiments.

In peripheral blood mononuclear cells from dialysis patients or healthy subjects, liquid chromatography–mass spectrometry identified 347 crotonylated proteins (Lin et al., 2020). A total of 345 proteins were differentially expressed between hemodialysis patients and controls, 93 of them upregulated and 252 downregulated. However, functional enrichment analysis did not show significant differences between upregulated and downregulated proteins and no direct relation was observed between Kcr ratio and levels of expression.

In summary, histone lysine crotonylation is a recently described posttranslational histone modification which, like previously described ones, has the potential to regulated gene expression. To date, its function in disease has been more extensively investigated in kidney injury. AKI and tubular cell stress in culture are characterized by increased global histone crotonylation. While it is conceivable that this is the end-result of differential crotonylation of many different genes, with very different impact on the expression of these genes, supplementing crotonate both increased kidney histone crotonylation and was nephroprotective and this was associated with potential beneficial changes in the expression of nephroprotective and proinflammatory genes. This opens the door to explore therapeutic strategies based on the modulation of histone crotonylation that, besides supplementing crotonate, may also include modulation of the microbiota and the use of HDAC inhibitors. In this regard the literature on the nephroprotective effects of HDAC inhibition should be re-examined from the point of view of their potential regulation of histone crotonylation. A detailed exploration of the role of histone crotonylation in kidney disease may facilitate the study of its impact in health and disease of other organs. Finally, the regulation and role of non-histone crotonylation remains essentially unexplored. A recent manuscript using a proteomics approach identified 155 upregulated and 198 downregulated crotonylated proteins in peripheral blood mononuclear cells from IgA nephropathy patients, of which only three were histones. Since only six patients were studied, whether these changes are linked to IgA nephropathy itself, to CKD or to other factors, remains unclear (Huang et al., 2012). In any case, this manuscript identifies the potential of a systems biology approach and eventual bioinformatics and machine learning analyses in advancing the field. Thus, a systems biology analysis of the impact of HDAC inhibitors in AKI may identify new biomarkers or therapeutic targets.

JM-M, MF-B, DM-S, MS-N and MR-O assessed the articles and their relevance to the above topics and wrote the manuscript. AO and AS supervised and drafted the manuscript and are the corresponding authors.

Sources of support: FIS/FEDER funds (PI15/00298, CP14/00133, PI16/02057, PI16/01900, PI18/01386, PI19/00588, PI19/00815, DTS18/00032, ERA-PerMed-JTC2018 (KIDNEY ATTACK AC18/00064 and PERSTIGAN AC18/00071), ISCIII-RETIC REDinREN RD016/0009), Sociedad Española de Nefrología, FRIAT, Comunidad de Madrid en Biomedicina B2017/BMD-3686 CIFRA2-CM. Salary support: ISCIII Miguel Servet and to AS and MS-N, ISCIII Sara Borrell to JM-M and Comunidad de Madrid (B2017/BMD-3686 CIFRA2-CM) to MF-B and DM-S. The authors declare no conflict of interest.

AS, AO, and M-SN have a patent on the use of crotonate to prevent or treat acute kidney injury.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.