Reduced methylation correlates with diabetic nephropathy risk in type 1 diabetes
Ishant Khurana, … , Per-Henrik Groop, Assam El-Osta
Ishant Khurana, … , Per-Henrik Groop, Assam El-Osta
Published January 12, 2023
Citation Information: J Clin Invest. 2023;133(4):e160959. https://doi.org/10.1172/JCI160959.
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Research Article Metabolism Nephrology

Reduced methylation correlates with diabetic nephropathy risk in type 1 diabetes

Abstract

Diabetic nephropathy (DN) is a polygenic disorder with few risk variants showing robust replication in large-scale genome-wide association studies. To understand the role of DNA methylation, it is important to have the prevailing genomic view to distinguish key sequence elements that influence gene expression. This is particularly challenging for DN because genome-wide methylation patterns are poorly defined. While methylation is known to alter gene expression, the importance of this causal relationship is obscured by array-based technologies since coverage outside promoter regions is low. To overcome these challenges, we performed methylation sequencing using leukocytes derived from participants of the Finnish Diabetic Nephropathy (FinnDiane) type 1 diabetes (T1D) study (n = 39) that was subsequently replicated in a larger validation cohort (n = 296). Gene body–related regions made up more than 60% of the methylation differences and emphasized the importance of methylation sequencing. We observed differentially methylated genes associated with DN in 3 independent T1D registries originating from Denmark (n = 445), Hong Kong (n = 107), and Thailand (n = 130). Reduced DNA methylation at CTCF and Pol2B sites was tightly connected with DN pathways that include insulin signaling, lipid metabolism, and fibrosis. To define the pathophysiological significance of these population findings, methylation indices were assessed in human renal cells such as podocytes and proximal convoluted tubule cells. The expression of core genes was associated with reduced methylation, elevated CTCF and Pol2B binding, and the activation of insulin-signaling phosphoproteins in hyperglycemic cells. These experimental observations also closely parallel methylation-mediated regulation in human macrophages and vascular endothelial cells.

Authors

Ishant Khurana, Harikrishnan Kaipananickal, Scott Maxwell, Sørine Birkelund, Anna Syreeni, Carol Forsblom, Jun Okabe, Mark Ziemann, Antony Kaspi, Haloom Rafehi, Anne Jørgensen, Keith Al-Hasani, Merlin C. Thomas, Guozhi Jiang, Andrea O.Y. Luk, Heung Man Lee, Yu Huang, Yotsapon Thewjitcharoen, Soontaree Nakasatien, Thep Himathongkam, Christopher Fogarty, Rachel Njeim, Assaad Eid, Tine Willum Hansen, Nete Tofte, Evy C. Ottesen, Ronald C.W. Ma, Juliana C.N. Chan, Mark E. Cooper, Peter Rossing, Per-Henrik Groop, Assam El-Osta

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Figure 7

Integration of FinnDiane methylation and renal epigenomic indices.

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Integration of FinnDiane methylation and renal epigenomic indices. (A) R...
(A) Renal cell types (podocytes and proximal convoluted tubule) assessed for DNA methylation mediated expression. (B) Reduced leukocyte methylation is a feature of hyperglycemia-induced podocyte changes. Comparison of differential methylation (~80,000 DMRs) in FinnDiane with human podocytes using rank-versus-rank analysis. Density plots show regions with gain (+) and loss (–) of DNA methylation in the FinnDiane discovery cohort (y axis) compared with human podocytes (x axis). Podocyte methylation sequencing data derived from 3 biological experiments (n = 3 per group) that were maintained in NG (physiological glucose control), NG + 5adC (physiological glucose including 3-day treatment with 5-aza-2′-deoxycytidine), HG (15 days high glucose), and HG + 5adC (15 days high glucose including 3-day treatment with 5adC). Similar pattern observed between differential methylation in FinnDiane versus human podocytes stimulated by HG and/or 5adC. (C) Diabetic nephropathy is associated with pathways regulated by DNA methylation in FinnDiane leukocytes and human podocytes. Annotated gene name; DNA methylation intensity - scaled (–2, relative methylation loss; +2, relative methylation gain), showing relative gene methylation of human podocytes maintained in NG, NG + 5adC, HG, and HG + 5adC. ChIP-seq data show MeCP2, CTCF, and Pol2B binding intensities relative to inputs (–1, low binding; +1, high binding). (D) mRNA expression levels associated with the progression of diabetic nephropathy. These genes are involved in pathways including insulin signaling, lipid metabolism, and integrin-cell interactions. Log2 scale — relative expression (–1, reduced; +1, elevated). (E) Profiling of phosphoprotein activation in human podocytes (relative to control, NG) using antibody array for the insulin signaling pathway. Proteins (219) were assessed using site-specific and phospho-specific antibodies (n = 2), NG, NG + 5adC, HG, and HG + 5adC. Relative protein activation is normalized to control (NG). Heatmap rows and columns are clustered using correlation distance and average linkage.