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DNA methylation aberrancies are found in autosomal dominant polycystic kidney disease (ADPKD), which suggests the methylome to be a promising therapeutic target. However, the impact of combining DNA methylation inhibitors (DNMTi) and ADPKD drugs in treating ADPKD and on disease-associated methylation patterns has not been fully explored. To test this, ADPKD drugs, metformin and tolvaptan (MT), were delivered in combination with DNMTi 5-aza-2′-deoxycytidine (Aza) to 2D or 3D cystic Pkd1 heterozygous renal epithelial cells (PKD1-Het cells) as free drugs or within nanoparticles to enable direct delivery for future in vivo applications. We found Aza synergizes with MT to reduce cell viability and cystic growth. Reduced representation bisulfite sequencing (RRBS) was performed across four groups: PBS, Free-Aza (Aza), Free-Aza+MT (F-MTAza), and Nanoparticle-Aza+MT (NP-MTAza). Global methylation patterns showed that while Aza alone induces a unimodal intermediate methylation landscape, Aza+MT recovers the bimodality reminiscent of somatic methylomes. Importantly, site-specific methylation changes associated with F-MTAza and NP-MTAza were largely conserved including hypomethylation at ADPKD-associated genes. Notably, we report hypomethylation of cancer-associated genes implicated in ADPKD pathogenesis as well as new target genes that may provide additional therapeutic effects. Overall, this study motivates future work to further elucidate the regulatory mechanisms of observed drug synergy and apply these combination therapies in vivo. 

Abstract Esophageal pathologies such as atresia and benign strictures often require surgical reconstruction with autologous tissues to restore organ continuity. Complications such as donor site morbidity and limited tissue availability have spurred the development of acellular grafts for esophageal tissue replacement. Acellular biomaterials for esophageal repair rely on the activation of intrinsic regenerative mechanisms to mediate de novo tissue formation at implantation sites. Previous research has identified signaling cascades involved in neoepithelial formation in a rat model of onlay esophagoplasty with acellular silk fibroin grafts, including phosphoinositide 3‐kinase (PI3K), and protein kinase B (Akt) signaling. However, it is currently unknown how these mechanisms are governed by DNA methylation (DNAme) during esophageal wound healing processes. Reduced‐representation bisulfite sequencing is performed to characterize temporal DNAme dynamics in host and regenerated tissues up to 1 week postimplantation. Overall, global hypermethylation is observed at postreconstruction timepoints and an inverse correlation between promoter DNAme and the expression levels of differentially expressed proteins during regeneration. Site‐specific hypomethylation targets genes associated with immune activation, while hypermethylation occurs within gene bodies encoding PI3K‐Akt signaling components during the tissue remodeling period. The data provide insight into the epigenetic mechanisms during esophageal regeneration following surgical repair with acellular grafts.