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. 2016 Feb;126(2):721-31.
doi: 10.1172/JCI82819. Epub 2016 Jan 5.

DNA methyltransferase inhibition restores erythropoietin production in fibrotic murine kidneys

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

DNA methyltransferase inhibition restores erythropoietin production in fibrotic murine kidneys

Yu-Ting Chang et al. J Clin Invest. 2016 Feb.

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.

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Figures

Figure 7
Figure 7. Aza restores EPO expression and ameliorates anemia in an adenine-induced kidney fibrosis model.
(A) Schema illustrating Aza or Veh treatment in mice fed with regular or adenine chow, as indicated in Supplemental Figure 9A. Phlebotomy was or was not performed 1 day before analyses at week 10. n = 12 per group. (B and C) Hematocrit, renal Epo expression, and plasma EPO levels of mice fed with regular or adenine chow and treated with Veh or Aza according to the schema in A. (D) Plasma levels of BUN and creatinine in mice fed with regular or adenine chow and treated with Veh or Aza according to the schema in A. (E) Renal expression of Acta2 and Col1a1 in mice fed with regular or adenine chow and treated with Veh or Aza according to the schema in A. One-way ANOVA was used for data analyses. †P < 0.01, ‡P < 0.001.
Figure 6
Figure 6. Aza restores EPO expression in fibrotic kidneys induced by UUO.
(A) Expression of Dnmt isoforms in CL and UUO kidneys after surgery. n = 10 per group. (B) Confocal images of DNMT1, DNMT3a, DNMT3b, and nidogen staining on kidney sections of Col1a1-GFPTg mice. Arrowheads highlight Col1a1-GFP+DNMT1+ or Col1a1-GFP+DNMT3a+ cells. Original magnification, ×400. Scale bar: 20 μm. (C) Schema illustrating Aza or Veh treatment in mice after UUO surgery. Phlebotomy was or was not performed 1 day before analyses at day 14 after UUO surgery. n = 10 per group. (D–F) Hematocrit, renal Epo expression, and plasma EPO levels in mice after UUO surgery and treatment with Veh or Aza according to the schema in C. One-way ANOVA was used for data analyses. *P < 0.05, †P < 0.01, ‡P < 0.001.
Figure 5
Figure 5. Aza restores EPO expression in myofibroblasts and TGF-β1–exposed pericytes.
(A) Schema showing CoCl2 stimulation of myofibroblasts after 3-day exposure to Aza or Veh and then 2-day drug withdrawal. (B) Western blot analysis showing the inhibitory effect of 500 nM Aza on DNMT1 expression of UUO kidney myofibroblasts. A representative blot of 4 independent experiments is shown. (C) Epo 5′-UTR methylation in myofibroblasts determined by MSP using the same method as in Figure 4, D and E. Myofibroblasts were analyzed at day 5, as described in A. Representative electrophoresis of 4 independent experiments is shown. (D) Annexin V apoptosis assay of myofibroblasts at day 3 and day 5, as described in A. n = 4 per group per time point. (E) Cell cycle analysis of myofibroblasts by measuring DNA content using propidium iodide staining at day 5, as described in A. The data were means of 4 independent experiments. (F) Expression of Epo, Phd3, and Vegfa in myofibroblasts with or without CoCl2 for 16 hours. Transient exposure to Aza was performed, as described in A. n = 4 per group. (G) Expression of Acta2 in myofibroblasts at day 5, as described in A. n = 4 per group. (H) Expression of Dnmt1, Dnmt3a, Dnmt3b, and Tgfb1 in kidney pericytes cultured in medium containing 5 ng/ml TGF-β1 or Veh for 24 hours. n = 4 per group. (I) Schema illustrating the culture of pericytes with or without TGF-β1 in the presence of 500 nM Aza or Veh. (J) Methylation of Epo 5′-UTR in pericytes determined by MSP at day 3, as described in I. Representative electrophoresis of 4 independent experiments is shown. (K) Expression of Epo, Phd3 and Vegfa in pericytes at day 3, as described in I. n = 4 per group. One-way ANOVA was used for analyses of data in D, F, H, and K, and Student’s t test was used for analyses of data in E and G. *P < 0.05, †P < 0.01, ‡P < 0.001.
Figure 4
Figure 4. 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.
Figure 3
Figure 3. Myofibroblast transition represses EPO.
(A) Hematocrit of UUO mice with or without phlebotomy 1 day before analyses at the indicated time points. n = 10 per group per time point. (B) Expression of Epo and Phd3 in CL and UUO kidneys. n = 10 per group per time point. (C) Expression of Epo, Vegfa, and Phd3 of Col1a1-GFP+PDGFRβ+ pericytes and myofibroblasts isolated from CL kidneys and kidneys 7 days after UUO surgery from Col1a1-GFPTg mice, respectively. n = 4 per cell group. (D) Epo expression of pericytes and myofibroblasts cultured in the presence of IOX2 for 24 hours. n = 4 per group. (E) Plasma levels of BUN and creatinine in mice fed with regular chow or chow containing 0.25% adenine for 21 days. n = 10 per group. (F–H) Hematocrit, renal Epo expression, and plasma EPO levels of mice fed with regular or adenine chows. n = 10 per group. (I) Epo expression of Col1a1-GFP+PDGFRβ+ pericytes and myofibroblasts isolated from kidneys of Col1a1-GFPTg mice fed with regular and adenine chows, respectively. n = 4 per cell group. 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, †P < 0.01, ‡P < 0.001.
Figure 2
Figure 2. HIF2α regulates EPO production in kidney pericytes.
(A) PCR products using kidney genomic DNA and genotyping primers for Hif2afl/fl mice. The knockout band was confirmed in Foxd1Cre/+ Hif2afl/fl mice. Foxd1+/+ Hif2afl/fl control mice only show the Hif2afl/fl band. (B) Analyses of hematocrit in Foxd1+/+ Hif2afl/fl and Foxd1Cre/+ Hif2afl/fl mice. n = 10 per group per time point. (C and D) Expression of renal Hif2a, Hif1a, and Epo and plasma EPO levels in 8-week-old adult mice. (E–G) Hematocrit, renal Epo expression, and plasma EPO levels in 8-week-old adult mice with and without phlebotomy. Student’s t test and 1-way ANOVA were used for analyses of data in B–D and E–G, respectively. *P < 0.05, †P < 0.01, ‡P < 0.001.
Figure 1
Figure 1. Col1a1-GFP+ pericytes are REPCs.
(A) Hematocrit (Hct) and plasma EPO concentrations and renal expression of Epo, Phd2, Phd3, and Vegfa normalized by Ubc in mice with and without phlebotomy (Con). Phlebotomy was performed 1 day before analysis. n = 5 per group. (B) Confocal images of kidney sections of EpoIRES-RFP/+ Col1a1-GFPTg mice. Arrowheads indicate EPO-RFP+Col1a1-GFP+ pericytes. T, renal tubules. Original magnification, ×400. Scale bar: 20 μm. (C) Expression of Epo, Phd2, Phd3, and Vegfa in Col1a1-GFP+PDGFRβ+ kidney pericytes isolated from Col1a1-GFPTg mice. n = 5 per group. (D) Fluorescent (left) and bright-field (right) images of primary cultures of live Col1a1-GFP+ kidney pericytes. Original magnification, ×400. Scale bar: 25 μm. (E) Epo expression and supernatant EPO concentration of Col1a1-GFP+ kidney pericytes cultured in chambers with normoxia (21% O2) or hypoxia (0.5% O2) for 24 hours. n = 4 per group. (F and G) Epo expression of Col1a1-GFP+ kidney pericytes cultured in the presence of CoCl2. n = 4 per group. (H) Epo expression of Col1a1-GFP+ kidney pericytes cultured in the presence of IOX2 for 24 hours. n = 4 per group. Student’s t test and 1-way ANOVA were used for analyses of data in A, C, and E and F–H, respectively. *P < 0.05, †P < 0.01, ‡P < 0.001.
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