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 9

Hyperglycemia influences the DDNs in proximal tubule cells and macrophages.

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Hyperglycemia influences the DDNs in proximal tubule cells and macrophag...
(A) Overview of experiments: culture conditions and experimental procedures used to assess core gene methylation and mRNA expression. Human proximal convoluted tubule cells and M1 macrophages (THP-1+ monocyte–derived) were cultured in physiological glucose conditions, high glucose (HG), and 5-aza-2′-deoxycytidine (5adC). (B) MTOR, RPTOR, IRS2, COL1A2, TXNRD1, LCAT, and SMPD3 were assessed using methyl–qPCR in PCT cells exposed to normal glucose (NG) for 15 days or NG for 15 days including 3 days with 5adC (NG + 5adC), HG for 15 days (HG), and HG including 3-days with 5adC (HG + 5adC) (n = 3). (C) mRNA levels of core genes assessed in PCT cells stimulated by chronic HG and 5adC. qRT-PCR data are shown relative to H3F3A (n = 3). (D) Macrophage differentiation from THP-1+ monocytes treated with phorbol-12-myristate-13-acetate (PMA) for 1 day and 15 days. Expression of macrophage-specific markers CD68, CD86, and TNFA (M1 macrophages) and CD163 and ARG1 (M2 macrophages) assessed by qRT-PCR. Data are shown relative to H3F3A (n = 3). (E) Methylation analysis of core genes in M1 macrophages (differentiated THP-1 day 15) exposed to HG and/or 5adC (n = 3). (F) mRNA levels of core genes assessed in M1 macrophages stimulated by chromic HG and 5adC. Data are shown relative to H3F3A. Significance was calculated using 2-tailed Student’s t test by comparing NG vs. HG, NG vs. NG + 5adC, and NG vs. HG + 5adC (B, C, E, and F) or by comparing undifferentiated vs. day 1 and undifferentiated vs. day 15 (D). *P < 0.05, **P < 0.01, ***P < 0.001. Error bars are SEM.