Nephropathic cystinosis is a rare lysosomal storage disorder presenting early in life with renal Fanconi syndrome that progressively leads to chronic kidney disease (CKD). The disease is caused by variants in the CTNS gene, which encodes the lysosomal cystine/H+ symporter cystinosin. Progressively, cystine and cystine crystals accumulate in all tissues, causing a multisystemic disease. To date, the only approved treatment for cystinosis is cysteamine, a sulfhydryl compound that reduces disulfide bonds, allowing cystine clearance from lysosomes. Cysteamine significantly slows the progression of kidney failure but, unfortunately, cannot prevent it, suggesting that cystine accumulation is not the sole mechanism driving kidney failure. Other proposed mechanisms include increased oxidative stress, mitochondrial function impairment, enhanced apoptosis, and abnormal autophagy (reviewed in Sur et al., 2024).

Epigenetic modifications, particularly DNA methylation (DNAm) at cytosine-phosphate-guanine (CpG) sites, have emerged as significant contributors to kidney parenchymal damage and CKD (Wanner and Bechtel-Walz, 2017). Large-scale studies have highlighted the importance of DNAm in preserving kidney health. For example, DNAm has been shown to explain a higher portion of kidney disease heritability than gene expression in high-throughput DNA methylation and transcriptomic profiling of 506 human kidneys (Liu et al., 2022). Similarly, meta-analysis of epigenome-wide association studies performed in ∼35,000 adults has identified multiple CpG sites with causal links to kidney function, further supporting the role of epigenetic regulation in kidney health and disease (Schlosser et al., 2021).

DNAm is a reversible process that modulates gene expression without modifying the DNA sequence. Specifically, DNA methylation in the promoter and in the first exon/intron regions can suppress gene expression by recruiting transcriptional repressors or by preventing binding of transcription factors (Anastasiadi et al., 2018; Bird, 1986).

Despite the growing understanding of these processes, the mechanisms underlying epigenetic dysregulation in kidney disease remain incompletely defined. Several lines of evidence support a link between the epigenome and cellular metabolism. In particular, recent data point to a critical crosstalk between oxidative stress, inflammation and epigenetic regulation, which may further contribute to the metabolic and inflammatory dysfunction observed in CKD (Liu et al., 2022; Stenvinkel et al., 2007). Since oxidative stress and inflammation have been shown to play a pathogenic role in cystinosis (Prencipe et al., 2014; Rossi et al., 2019; Vaisbich et al., 2011), we have hypothesized that epigenetic dysregulation might also contribute to CKD progression. To investigate this hypothesis, we conducted a comprehensive methylation analysis on kidneys harvested from Ctns ^−/− mice, a well-established model for cystinosis (Nevo et al., 2010).

The Ctns ^−/− mouse is a well-established model for kidney disease in cystinosis (Nevo et al., 2010). Mice typically develop evidence of kidney damage, including tubular atrophy, inflammation, and interstitial fibrosis around 6 months of age (Nevo et al., 2010; Rossi et al., 2024). In this study, we have performed whole-genome DNAm profiling in 6-month-old kidneys to explore the potential role of epigenetic alterations in cystinosis-related kidney disease. Our results show significant epigenetic differences between Ctns ^−/− and WT mice, with a pronounced hypermethylation profile in cystinotic kidneys, even at this early stage of renal parenchymal damage. These methylation changes were primarily found in genes and pathways crucial for kidney function, particularly those related to the physiology of proximal tubules. Importantly, these methylation alterations correlated with changes in gene expression, as validated by qPCR analyses performed on key selected genes.

DNAm is a key epigenetic mechanism, and abnormal changes have been associated with a wide range of diseases, including cancer, and more recently, kidney diseases (Bechtel et al., 2010; Ko et al., 2013; Larkin et al., 2018; Marumo et al., 2015; Sagy et al., 2024; Smyth et al., 2014; Tampe et al., 2017; Tampe et al., 2015; Tampe et al., 2014; Yan et al., 2024). In our study, we found both hypermethylation (85% of DMP) and hypomethylation (15% of DMP) in cystinotic kidneys compared to WT kidneys. Both alterations, hypermethylation and hypomethylation, have potential effects in gene expression regulation and have been described in a wide range of pathologies spanning from cancer to neurodegenerative diseases (Ehrlich, 2009; Lu et al., 2013). Interestingly, our genome-wide methylation analysis showed that in Ctns ^−/− mice kidneys the majority of differentially methylated CpG sites were located within gene bodies (around 66%), while approximately 11% of DMP was found in promoter and enhancer regions. Previous studies have focused on CpG islands in promoter regions and their role in regulating gene expression (Fernandez et al., 2012; Nagae et al., 2011), but increasingly more studies are focusing on differentially methylated regions (DMRs) in gene bodies (Anastasiadi et al., 2018; Lokk et al., 2014), where fully methylated CpGs are more often reported (Bontha et al., 2017; Illingworth and Bird, 2009; Ko et al., 2013). Generally, promoter hypermethylation leads to heterochromatin formation, resulting in tightly packed DNA, reduced transcription factor accessibility, and gene silencing; DNA hypomethylation is often associated with gene activation or genomic instability (Illingworth and Bird, 2009). Conversely, gene-body methylation has been reported to positively correlate with gene expression, although it can also interfere with transcription elongation (Lokk et al., 2014). Increasingly, it is becoming clear that the relationship between gene-body methylation and mRNA expression levels is more nuanced and complex than initially thought. In our study, we cannot draw broad conclusions on the functional implications of gene-body hypermethylation in Ctns ^−/− kidneys based solely on location data. Nevertheless, when we sampled gene expression, we observed that hypermethylated regions, whether in gene bodies or promoter regions, were consistently associated with reduced mRNA levels. These findings underscore the importance of integrating DNA methylation and gene expression data and performing functional analyses to understand the precise molecular mechanisms underlying cystinosis-related kidney dysfunction.

Pathway enrichment analysis highlighted a significant enrichment of methylation changes in the “Kidney Tubule Cell” cluster, further emphasizing the critical role of tubular epithelial cells in the pathogenesis of kidney damage in cystinosis (Ivanova et al., 2023; Sur et al., 2024). Additionally, functional annotation revealed that many of the differentially methylated genes were predominantly involved in pathways related to “Transport of small molecules”, a process essential for maintaining renal function and electrolyte balance (Lewis et al., 2021). Strikingly, we found that 93% (55 out of 57) of differentially methylated Slc genes were hypermethylated. Accordingly, we found that key transporters such as Slc7a7, Slc4a4, as well as Cubn and Aqp1 were not only hypermethylated but also significantly downregulated in cystinotic kidneys, suggesting that epigenetic silencing of transport-related genes could exacerbate tubular dysfunction. Similarly, in the kidneys of Ctns ^−/− mice, we found hypermethylation and reduced mRNA expression levels of transcription factors Hnf1a and Hnf4a, both critical for proximal tubule differentiation and function (Marable et al., 2018), further supporting the role of epigenetic modifications in disrupting tubular epithelial integrity. Indeed, loss of Hnf1a and Hnf4a expression has been implicated in the development of proximal tubular dysfunction (Marable et al., 2018), resulting in glycosuria and polyuria, which are hallmark features of cystinosis. Of note, Marumo et al. (2015) have observed that diabetes induces aberrant DNAm changes in proximal tubules, in particular in Hnf4a gene. Furthermore, recent findings emphasize the central role of DNAm in kidney-specific expression of amino acid transporters and of their master regulator, Hnf1a (Kikuchi et al., 2010). Consistently, our unpublished transcriptomic analyses of cortical kidney sections from 12-month-old mice revealed that 145 Slc genes were differentially expressed in cystinotic kidneys compared to WT mice, with 115 genes (79%) downregulated (data not shown). These findings strongly suggest that epigenetic dysregulation of transcription factors and transport-related genes plays a pivotal role in the pathogenesis and progression of cystinosis.

While the precise mechanisms underlying epigenetic dysregulation in kidney diseases remain unclear, emerging evidence points to oxidative stress and inflammation as key contributors (Elmonem et al., 2022; Ingrosso and Perna, 2020). Oxidative stress, a known driver of cystinosis-related tubular damage, has been shown to modulate DNAm patterns through alterations in methylation enzymes like DNMTs and TET proteins (Tampe et al., 2014). This interplay could amplify the hypermethylation observed in genes crucial for tubular cell function. Similarly, chronic inflammation, which characterizes cystinotic kidneys, can induce epigenetic changes that perpetuate inflammatory signaling and tubular injury. On these basis, it would be interesting to investigate whether interventions that have proven effective in preventing or delaying the progression of cystinosis, such as the ketogenic diet (Bellomo et al., 2024) or flavonoids (De Leo et al., 2024; De Leo et al., 2023), both known for their antioxidant and anti-inflammatory properties, exert at least in part, their effects by modulating the epigenetic landscape of cystinotic kidneys in mice.

The reversibility of DNA methylation represents an intriguing therapeutic opportunity for cystinosis. In our study, treatment of cystinotic ciPTECs with the demethylating agent decitabine successfully increased the expression of key transporters, including SLC7A7, AQP1, and CUBN. It is noteworthy that these genes were already transcriptionally active in HD cells, likely making them more responsive to upregulation upon treatment. Decitabine has been FDA-approved for the treatment of myelodysplastic syndrome and acute myeloid leukemia (Derissen et al., 2013) and, currently, several clinical trials on cancer are testing the efficacy of demethylating drugs (Wanner and Bechtel-Walz, 2017). Moreover, epidrugs are also effective to slow down the progression of renal fibrosis and inflammation in animals with progressive CKD (Tampe et al., 2017; Wanner and Bechtel-Walz, 2017) and decitabine treatment has been successfully used in mouse models of diabetic nephropathy (Larkin et al., 2018; Zhang et al., 2017). These findings highlight the therapeutic potential of epigenetic modulation to counteract gene silencing and improve tubular function. However, confirming the effect at the protein level and assessing downstream functional parameters would be essential to establish the real impact on cell physiology. Furthermore, an in vivo study using cystinotic mice treated with decitabine would be crucial to understand whether and how demethylation concretely affects disease pathogenesis.

In conclusion, this study provides compelling evidence that epigenetic alterations, particularly hypermethylation, contribute to cystinosis-related renal dysfunction. The identification of differentially methylated genes and pathways, especially those involved in tubular transport and transcriptional regulation, offers new insights into the molecular mechanisms driving disease progression. These findings pave the way for future research into the therapeutic potential of targeting DNAm to mitigate kidney damage in cystinosis. They also show that integrating epigenomic, transcriptomic, and proteomic data is essential to develop a comprehensive understanding of cystinosis-related kidney disease and to identify novel therapeutic targets for intervention.