DNA methyltransferase inhibition restores erythropoietin production in fibrotic murine kidneys
Yu-Ting Chang, … , Yung-Ming Chen, Shuei-Liong Lin
Yu-Ting Chang, … , Yung-Ming Chen, Shuei-Liong Lin
Published January 5, 2016
Citation Information: J Clin Invest. 2016;126(2):721-731. https://doi.org/10.1172/JCI82819.
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Research Article Nephrology

DNA methyltransferase inhibition restores erythropoietin production in fibrotic murine kidneys

Abstract

Renal erythropoietin-producing cells (REPCs) remain in the kidneys of patients with chronic kidney disease, but these cells do not produce sufficient erythropoietin in response to hypoxic stimuli. Treatment with HIF stabilizers rescues erythropoietin production in these cells, but the mechanisms underlying the decreased response of REPCs in fibrotic kidneys to anemic stimulation remain elusive. Here, we show that fibroblast-like FOXD1+ progenitor-derived kidney pericytes, which are characterized by the expression of α1 type I collagen and PDGFRβ, produce erythropoietin through HIF2α regulation but that production is repressed when these cells differentiate into myofibroblasts. DNA methyltransferases and erythropoietin hypermethylation are upregulated in myofibroblasts. Exposure of myofibroblasts to nanomolar concentrations of the demethylating agent 5-azacytidine increased basal expression and hypoxic induction of erythropoietin. Mechanistically, the profibrotic factor TGF-β1 induced hypermethylation and repression of erythropoietin in pericytes; these effects were prevented by 5-azacytidine treatment. These findings shed light on the molecular mechanisms underlying erythropoietin repression in kidney myofibroblasts and demonstrate that clinically relevant, nontoxic doses of 5-azacytidine can restore erythropoietin production and ameliorate anemia in the setting of kidney fibrosis in mice.

Authors

Yu-Ting Chang, Ching-Chin Yang, Szu-Yu Pan, Yu-Hsiang Chou, Fan-Chi Chang, Chun-Fu Lai, Ming-Hsuan Tsai, Huan-Lun Hsu, Ching-Hung Lin, Wen-Chih Chiang, Ming-Shiou Wu, Tzong-Shinn Chu, Yung-Ming Chen, Shuei-Liong Lin

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Figure 4

Hypermethylation of EPO 5′-regulatory elements in kidney myofibroblasts.

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Hypermethylation of EPO 5′-regulatory elements in kidney myofibroblasts....
(A) Schema of COBRA, illustrating the locations of PCR primers (forward and reverse for amplifying bisulfite-converted genomic DNA and recognition sites of the BstUI restriction enzyme in the Epo promoter and 5′-UTR. (B) Representative COBRA showing the electrophoresis of PCR products with (+) or without (–) BstUI digestion from 3 independent experiments. Bisulfite-converted genomic DNA was prepared from pericytes and myofibroblasts isolated from normal kidneys and kidneys 14 days after UUO surgery from Col1a1-GFPTg mice, respectively. Meth, methylated; Unmeth, unmethylated controls. (C) BGS of Epo promoter and 5′-UTR of pericytes and myofibroblasts. Each box represents the bisulfite genomic sequence of the indicated cell isolated from one Col1a1-GFPTg mouse; each row represents a single sequenced clone (4 clones from each mouse); and each dot represents a single CpG. (D) Schema of MSP illustrating the locations of primers for unmethylated and methylated Epo 5′-UTR. (E) Representative electrophoresis of MSP using primers for unmethylated (U) and methylated (M) Epo 5′-UTR. (F) The percentage of Epo 5′-UTR methylation in pericytes and myofibroblasts determined by the densitometric analyses of MSP products. n = 4 per cell group. (G) BGS of Epo distal HRE+ 5′-enhancer of pericytes and myofibroblasts. Boxes, rows, and dots are as defined in the legend for C. The sequences of PCR primers are shown in Supplemental Table 2. One-way ANOVA was used for analyses of data in A–C and E–H, and Student’s t test was used for analyses of data in D. *P < 0.05.