Epigenetic Mechanisms Involved in Cisplatin-Induced Nephrotoxicity: An Update

Cisplatin is an antineoplastic drug used for the treatment of many solid tumors. Among its various side effects, nephrotoxicity is the most detrimental. In recent years, epigenetic regulation has emerged as a modulatory mechanism of cisplatin-induced nephrotoxicity, involving non-coding RNAs, DNA methylation and histone modifications. These epigenetic marks alter different signaling pathways leading to damage and cell death. In this review, we describe how different epigenetic modifications alter different pathways leading to cell death by apoptosis, autophagy, necroptosis, among others. The study of epigenetic regulation is still under development, and much research remains to fully determine the epigenetic mechanisms underlying cell death, which will allow leading new strategies for the diagnosis and therapy of this disease.


Introduction
Globally, cancer is the second leading cause of death. In 2018, 18.1 million people worldwide had cancer, and 9.6 million died from this disease [1]. Cisplatin [cisdiamminedichloroplatinum (II), CDDP] is a well-known chemotherapeutic drug used for the treatment of numerous human cancer in solid organs, including head and neck [2], testis [3], small cells [4] and non-small cells [5] lung cancer, ovarian [6], cervical [7] and bladder [8]. Regarding the treatment of cancer cells, CDDP is potentially accompanied by some side effects such as ototoxicity, gastrotoxicity, neurotoxicity, myelosuppression, and allergic reactions [9]. Still, the main limiting side effect of CDDP use is nephrotoxicity [10]. Nephrotoxicity is defined as the rapid deterioration in kidney function due to the toxic effect of drugs, medications, and/or chemicals [11]. Cisplatin-induced-nephrotoxicity can occur in several ways, but the most severe and common is acute kidney injury, whose incidence after a single dose of CDDP fluctuates between 20-30% in patients who receive an initial dose of 50-100 mg/m 2 [9]. Cisplatin is concentrated and reabsorbed by tubular renal cells (5 times more than in the blood), which triggers a rapid decrease in renal function [12], resulting in morphological changes and altered signaling pathways, such as vacuolation, mitochondrial dysfunction, cell cycle arrest and apoptosis [13,14]. The diagnosis of kidney damage is carried out using standard biomarkers, such as serum creatinine (sCr) and blood urea nitrogen (BUN). However, their sensitivity and specificity are very low, and values are altered when significant kidney damage already exists [15].
In recent years, several studies showed that epigenetic processes are involved in cisplatin-induced nephrotoxicity. Wu and Morris [16] defined epigenetic as the "study of major MAPKs: extracellular signal-regulated kinase (ERK), p38, and c-JUN N-terminal kinase (JNK) [35,41]. This induces oxidative stress, activating TNF-α and the consequent inflammatory response during CDDP-induced nephrotoxicity [42]. The augmented expression of ERK1/2 by CDDP treatment also activates NF-κβ and p53 signaling [43]. TNF-α increases tumor necrosis factors 1 and 2, TNFR1 and TNFR2, triggering the extrinsic apoptosis pathway through the activation of caspase-8, which will eventually lead to caspase-3 activation [9]. Cisplatin also regulates p21; its up-regulation protects from nephrotoxic damage [21]. Oxidative stress is a key factor contributing to nephrotoxicity since CDDP can react with glutathione, decreasing non-enzymatic (GSH and NADPH) and enzymatic (SOD, CAT, GPx, among others) antioxidant defense mechanisms [44]. Cisplatin also affects the mitochondrial respiratory complexes, leading to the inhibition of complexes I to IV of the respiratory chain and decreasing intracellular ATP levels [45]. In addition, CDDP also reduces the number of mitochondria in normal renal cells [43]. Finally, CDDP causes the activation of an adaptative program in the endoplasmic reticulum (ER) known as unfolded protein response (UPR), which inositol-requiring enzyme-1 (IRE1), double-stranded RNA-activated protein kinase-like ER kinase (PERK), and activating transcription factor-6 (ATF6) are dissociated from ER chaperone GRP78, activating signal transduction to inhibit protein translation [46]. Therefore, cisplatin-induced nephrotoxicity is a multifactorial and complex process involving multiple signaling pathways that in turn, may also be regulated by epigenetic mechanisms.

DNA Methylation Role in Cisplatin-Induced Renal Dysfunction
DNA methylation is an epigenetic modification involving the covalent addition of a methyl group to carbon 5 of a cytosine at CpG dinucleotides through the action of DNA methyltransferase enzymes (DNMTs) [47], which include DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L, contributing to hypermethylated (high methylation levels) or hypomethylated (low methylation levels) states of genes or genomic regions containing CpG residues [48]. In mammals, 70-80% of CpG dinucleotides are methylated, whereas CpG islands in promoter regions of genes are demethylated, allowing transcription to proceed [49]. Therefore, the main function of DNA methylation is the regulation of transcription. Thus, hypomethylation in the promoter region correlates with gene activation by increased access for transcription factors, whereas hypermethylation in the promoter region results in loss of gene expression. Studies associating different DNA methylation patterns with CDDP-induced nephrotoxicity are scarce. Reports show that treatment of tubular cells with 5-aza-2 -deoxycytidine (5-AZA), a DNA methylation inhibitor, increases CDDP-induced apoptosis [50,51]. In addition, IRF8, a pro-apoptotic factor, is hypomethylated and induced after CDDP treatment, contributing to renal tubular cell apoptosis ( Figure 1) [50]. In conjunction with 5-AZA, cisplatin decreases the expression of DNMT1, a maintenance DNMT, attenuating CDDP-induced nephrotoxicity [52]. It is recognized that OCT2, a transporter involved in organic cation transport, can transport CDDP to proximal tubules [53]. Based on this, studies show that methylation of the promoter region of OCT2 dramatically reduces the transcriptional activity of this transporter [54], which may contribute to increased toxicity.

Cisplatin Nephrotoxicity and Histone Modifications
The dynamic changes in chromatin structure, which allow its decondensation and remodeling, are processes necessary for gene transcription, DNA repair, and replication, carried out by post-translational modifications in the different histones [55]. Histones are the basic structural proteins of nucleosomes, which bind to double-helical DNA, forming the DNA-histone complex [47]. There are several types of histones, such as linker histones (H1 and H5) and core histones (H2A, H2B, H3, and H4), whose function is associated with DNA packaging [49], where CDDP can easily react with H1 at methionine and glutamate residues, forming tertiary complexes that prevent DNA repair and increase sensitivity to CDDP [56]. Histone modifications occur predominantly at the N-terminal end, changing chromatin structure, positively or negatively affecting gene expression [49]. There are at least 8 histone modifications, but the most studied include histone acetylation and methylation, mostly in lysine and arginine residues.
On the other hand, histone acetylation involves adding an acetyl group to the lysine residue of core histones by a group of enzymes called histone acetyltransferases (HATs) [47]. This promotes open chromatin because a negative charge is added to the positively charged lysines, reducing the strong DNA-histone interaction, and thus, gene expression is activated [58]. One member of HATs is p300. This HAT can directly bind to transcription factors, such as p53 or NF-κB, and regulate their activities by acetylation [59,60]. Cisplatin

Cisplatin Nephrotoxicity and Histone Modifications
The dynamic changes in chromatin structure, which allow its decondensation and remodeling, are processes necessary for gene transcription, DNA repair, and replication, carried out by post-translational modifications in the different histones [55]. Histones are the basic structural proteins of nucleosomes, which bind to double-helical DNA, forming the DNA-histone complex [47]. There are several types of histones, such as linker histones (H1 and H5) and core histones (H2A, H2B, H3, and H4), whose function is associated with DNA packaging [49], where CDDP can easily react with H1 at methionine and glutamate residues, forming tertiary complexes that prevent DNA repair and increase sensitivity to CDDP [56]. Histone modifications occur predominantly at the N-terminal end, changing chromatin structure, positively or negatively affecting gene expression [49]. There are at least 8 histone modifications, but the most studied include histone acetylation and methylation, mostly in lysine and arginine residues.
On the other hand, histone acetylation involves adding an acetyl group to the lysine residue of core histones by a group of enzymes called histone acetyltransferases (HATs) [47]. This promotes open chromatin because a negative charge is added to the positively charged lysines, reducing the strong DNA-histone interaction, and thus, gene expression is activated [58]. One member of HATs is p300. This HAT can directly bind to transcription factors, such as p53 or NF-κB, and regulate their activities by acetylation [59,60]. Cisplatin induces p300 activation, acetylating lysines 18, 27, and 9 on H3, triggering functional and histological injury, increasing oxidative stress, inflammation, and apoptosis ( Figure 2) [61]. One strategy to decrease CDDP-induced damage has been the inhibition of p300 by garcinol, attenuating the oxidative stress, inflammation, and apoptosis, by reducing the acetylation of the p65 subunit of NF-κB [61].

Involvement of Non-Coding RNAs in Cisplatin Nephrotoxicity
Less than 2% of the human genome is transcribed into RNA to be translated into proteins. The remaining 98% is transcribed into a class of RNA that is not translated into proteins but plays a critical role in epigenetics. These molecules are called non-coding RNAs (ncRNAs) [47]. They regulate gene expression under physiological and pathological conditions, acting at various stages in protein synthesis at both transcriptional and post-transcriptional levels [80]. Non-coding RNAs are further divided into two main groups, small ncRNAs (shorter than 200 bp) and long ncRNA (lncRNAs; longer than 200 bp).
Out of small ncRNAs, microRNAs (miRs) have been by far the most extensively studied. They are a class of short-chain, linear, approximately 21-25 nucleotides long that negatively regulate gene targets at the post-transcriptional level by perfect complementarity of their "seed" region to 3′-UTR of its target mRNA, inducing their degradation. If there is a mismatch or imperfect complementarity, it results in translational repression [49]. The latest release of the miRbase database (v22) contains 2654 human mature miRs sequences [81], ratifying their importance on gene expression regulation.

Involvement of Non-Coding RNAs in Cisplatin Nephrotoxicity
Less than 2% of the human genome is transcribed into RNA to be translated into proteins. The remaining 98% is transcribed into a class of RNA that is not translated into proteins but plays a critical role in epigenetics. These molecules are called non-coding RNAs (ncRNAs) [47]. They regulate gene expression under physiological and pathological conditions, acting at various stages in protein synthesis at both transcriptional and posttranscriptional levels [80]. Non-coding RNAs are further divided into two main groups, small ncRNAs (shorter than 200 bp) and long ncRNA (lncRNAs; longer than 200 bp).
Out of small ncRNAs, microRNAs (miRs) have been by far the most extensively studied. They are a class of short-chain, linear, approximately 21-25 nucleotides long that negatively regulate gene targets at the post-transcriptional level by perfect complementarity of their "seed" region to 3 -UTR of its target mRNA, inducing their degradation. If there is a mismatch or imperfect complementarity, it results in translational repression [49]. The latest release of the miRbase database (v22) contains 2654 human mature miRs sequences [81], ratifying their importance on gene expression regulation.

Potential Challenges and Future Directions
Epigenetics is an active research field concerning cisplatin-induced nephrotoxicity. So far, studies show that the mechanisms involved play a significant role in the pathogenesis of this condition. However, little is known about the interaction between different epigenetic marks. Nonetheless, crosstalk between epigenetic modifications does exist in this pathology. For example, during CDDP-induced nephrotoxicity, lncRNA LRNA9884 increases NF-κB expression [103]. On the other hand, miR-34 [21,36], miR-192-5p [24] and, miR-449 [32] activate p53. Both NF-κB and p53 up-regulate miR-375 [85], inducing apoptosis in renal proximal tubular cells. However, inhibiting p300 attenuates this adverse effect due to reduced acetylation of NF-κB [61]. In the same way, overexpression of SIRT1 and SIRT6 inhibits p53 signaling, reducing cisplatin-induced damage [43,74]. Clearly, there are many gaps regarding epigenetic modulation in the pathogenesis of this condition, especially in the cross-talking for these mechanisms. Understanding the way that DNA methylation, histone modifications, and ncRNAs underlie this disease will provide us with the opportunity to discover new biomarkers for early diagnosis, as well as the establishment of novel therapies or treatments aiming to decrease the incidence of cisplatin-induced nephrotoxicity. Although autophagy is generally activated under conditions of nutrient deprivation, it has also been implicated in physiological (development, differentiation) and pathological (neurodegenerative diseases, stress, infection, cancer) processes [86]. Based on this, it has been proven that CDDP also modulates the autophagy process in renal tubular cells [87,88], although the epigenetic mechanisms underlying its occurrence have been poorly studied. However, it is known that CDDP modulates MAPK/ERK1/2 signaling pathway through miR-146b up-regulation [89]. Enhanced mir-146b levels directly impact the expression of mTOR, an autophagy initiation regulatory kinase, which is in turn mediated by miR-199a up-regulation [38] and miR-26b down-regulation [90]. This allows the interaction of Atg13, FIPP200, and ULK1/2 [91], the last one being regulated by miR-141 repression [92]. The Atg13/FIPP200/ULK composite interacts with the PI3K class III complex, composed of several molecules, including Beclin-1, which recruit several ATG proteins, allowing the phagophore formation [91], and thus, starting the autophagy process.
Cisplatin also modulates cellular oxidative stress by regulating NRF2 [93], a transcription factor that regulates the expression of genes encoding antioxidant, anti-inflammatory, and detoxifying proteins [94]. Under normal conditions, NRF2 is located in the cytoplasm, negatively controlled by KEAP1; whereas, under oxidative stress conditions, KEAP1 releases NRF2, which can be translocated to the nucleus and, in this way, binds to antioxidant response elements (ARE), resulting in the transcription of antioxidant genes [95]. Some miRs have been shown to modulate KEAP1/NRF2 interaction, such as miR-192-5p [24] and miR-140-5p [93]. In addition to this, CDDP also alters the redox state, by decreasing the antioxidant activity of GSH, GSH-Px, and SOD [37].
Necroptosis is a programmed form of necrotic cell death considered passive cell death through regulated cell signaling pathways [96]. RIPK1, RIPK3, and MLKL are the main molecules involved in the necroptosis process. Necroptosis is triggered when TNFR1 is activated by TNF-α, resulting in complex I formation. Subsequently, RIPK1, TRADD, and FADD are recruited. Once RIPK1 is activated, its interaction with RIPK3 occurs, forming complex IIb/necrosome, which mediates the phosphorylation of MLKL, whose translocation to the plasma membrane promotes necroptosis by disrupting plasma membrane integrity [97]. Some studies in renal tubular cells have demonstrated the involvement of miR-500a-3p in RIPK1, RIPK3, and MLKL regulation when these cells are treated with CDDP [98,99]. On the other hand, RIPK1 ubiquitination results in NF-κB activation, mediating the survival and production of multiple inflammatory cytokines, which are also regulated by several miRs when these cells are exposed to CDDP. Thus, IL-6 is mediated by miR-494 [15] and miR-34a [37]; IL-8 by miR-500a-3p [99]; and finally, IL-1β by miR-155 [100] and miR-34a [37].
Circular RNAs (circRNAs) are single-stranded, covalently locked transcripts produced from a precursor mRNA [105]. These circRNAs can act as sponges for miRs, resulting in increased expression [49]. In this way, circ-0114427 attracts miR-494 and interacts with it, increasing ATF3 expression and reducing inflammatory cytokines, thus exerting a nephroprotective role against CDDP damage [15].      PTECs: Proximal Tubular Epithelial Cells.

Potential Challenges and Future Directions
Epigenetics is an active research field concerning cisplatin-induced nephrotoxicity. So far, studies show that the mechanisms involved play a significant role in the pathogenesis of this condition. However, little is known about the interaction between different epigenetic marks. Nonetheless, crosstalk between epigenetic modifications does exist in this pathology. For example, during CDDP-induced nephrotoxicity, lncRNA LRNA9884 increases NF-κB expression [103]. On the other hand, miR-34 [21,36], miR-192-5p [24] and, miR-449 [32] activate p53. Both NF-κB and p53 up-regulate miR-375 [85], inducing apoptosis in renal proximal tubular cells. However, inhibiting p300 attenuates this adverse effect due to reduced acetylation of NF-κB [61]. In the same way, overexpression of SIRT1 and SIRT6 inhibits p53 signaling, reducing cisplatin-induced damage [43,74]. Clearly, there are many gaps regarding epigenetic modulation in the pathogenesis of this condition, especially in the cross-talking for these mechanisms. Understanding the way that DNA methylation, histone modifications, and ncRNAs underlie this disease will provide us with the opportunity to discover new biomarkers for early diagnosis, as well as the establishment of novel therapies or treatments aiming to decrease the incidence of cisplatin-induced nephrotoxicity.

Conclusions
Nephrotoxicity is the most lethal side effect of cisplatin treatment. During the last few years, rapid progress has been made in understanding the contribution of epigenetic mechanisms underlying nephrotoxicity to be used as potential biomarkers for early diagnosis and/or to employ different therapeutic strategies. These studies demonstrate the complexity of the interactions of epigenetic modifications, which have provided a better understanding of the epigenetic regulation of CDDP-induced nephrotoxicity. Continued research in this area will provide new therapeutic targets and early, sensitive, and specific diagnostic biomarkers to detect CDDP-induced nephrotoxicity.