Aberrant proximal tubule DNA methylation underlies phenotypic changes related to kidney dysfunction in patients with diabetes

Epigenetic mechanisms are considered to contribute to diabetic nephropathy by maintaining memory of poor glycemic control during the early stages of diabetes. However, DNA methylation changes in the human kidney are poorly characterized, because of the lack of cell type-specific analysis. We examined DNA methylation in proximal tubules purified from diabetic nephropathy patients and identified differentially methylated CpG sites, given the critical role of proximal tubules in the kidney injury. Hypermethylation was observed at CpG sites annotated to genes responsible for proximal tubule functions, including gluconeogenesis, nicotinamide adenine dinucleotide synthesis, transporters of glucose, water, phosphate, and drugs, in diabetic kidneys, while genes involved in oxidative stress and the cytoskeleton exhibited demethylation. Methylation levels of CpG sites annotated to ACTN1, BCAR1, MYH9, UBE4B, AFMID, TRAF2, TXNIP, FOXO3, and HNF4A were correlated with the estimated glomerular filtration rate, while methylation of the CpG site in RUNX1 was associated with interstitial fibrosis and tubular atrophy. Hypermethylation of G6PC and HNF4A was accompanied by decreased expression in diabetic kidneys. Proximal tubule-specific hypomethylation of metabolic genes related to HNF4A observed in control kidneys was compromised in diabetic kidneys, suggesting a role for aberrant DNA methylation in the dedifferentiation process. Multiple genes with aberrant DNA methylation in diabetes overlapped genes with altered expressions in maladaptive proximal tubule cells, including transcription factors PPARA and RREB1. In conclusion, DNA methylation derangement in the proximal tubules of patients with diabetes may drive phenotypic changes, characterized by inflammatory and fibrotic features, along with impaired function in metabolism and transport. DNA was extracted from 15 samples of normal micro-dissected proximal tubules and 18 samples of micro-dissected proximal tubules obtained from patients with diabetic nephropathy by NucleoSpin Tissue XS (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany). DNA was then subjected to bisulfite conversion using the EZ DNA Methylation Gold Kit (Zymo Research, Irvine, CA). Genome-wide DNA methylation analysis was performed using the Illumina Infinium MethylationEPIC Kit (Illumina, San Diego, CA) using the iScan reader (Illumina). The data were assembled using the GenomeStudio methylation software (Illumina).